Plants which synthesize a modified starch, process for the production thereof and modified starch

ABSTRACT

Nucleic acid molecules are described encoding a starch granule-bound protein as well as methods and recombinant DNA molecules for the production of transgenic plant cells and plants synthesizing a modified starch with modified viscosity properties and a modified phosphate content. Moreover, the plant cells and plants resulting from those methods as well as the starch obtainable therefrom are described.

This application is a continuation of international applicationPCT/EP96/04109, filed Sep. 19, 1996, which international applicationdesignated the United States.

SUMMARY OF THE INVENTION

The present invention relates to nucleic acid molecules encoding astarch granule-bound protein as well as to methods and recombinant DNAmolecules for the production of transgenic plant cells and plantssynthesizing a modified starch with modified properties of viscosity anda modified phosphate content. The invention also relates to thetransgenic plant cells and plants resulting from these methods and tothe starch obtainable from the transgenic plant cells and plants.

BACKGROUND OF THE INVENTION

The polysaccharide starch, which constitutes one of the most importantstorage substances in plants, is not only used in the area of foodstuffsbut also plays a significant role as a regenerative material in themanufacturing of industrial products. In order to enable the use of thisraw material in as many areas as possible, it is necessary to obtain alarge variety of substances as well as to adapt these substances to thevarying demands of the processing industry.

Although starch consists of a chemically homogeneous basic component,namely glucose, it does not constitute a homogeneous raw material. It israther a complex mixture of various types of molecules which differ fromeach other in their degree of polymerization and in the degree ofbranching of the glucose chains. One differentiates particularly betweenamylose-starch, a basically non-branched polymer made up ofα-1,4-glycosidically branched glucose molecules, and amylopectin-starchwhich in turn is a mixture of more or less heavily branched glucosechains. The branching results from the occurrence of α-1,6-glycosidicinterlinkings.

The molecular structure of starch which is mainly determined by itsdegree of branching, the amylose/amylopectin ratio, the averagechain-length and the occurrence of phosphate groups is significant forimportant functional properties of starch or, respectively, its aqueoussolutions. Important functional properties are for example solubility ofthe starch, tendency to retrogradation, capability of film formation,viscosity, colour stability, pastification properties, i.e. binding andgluing properties, as well as cold resistance. The starch granule sizemay also be significant for the various uses. The production of starchwith a high amylose content is particularly significant. Furthermore,modified starch contained in plant cells may, under certain conditions,favorably alter the behavior of the plant cell. For example, it would bepossible to decrease the starch degradation during the storage of thestarch-containing organs such as seeds and tubers prior to their furtherprocessing by, for example, starch extraction. Moreover, there is someinterest in producing modified starches which would render plant cellsand plant organs containing this starch more suitable for furtherprocessing, such as for the production of popcorn or corn flakes frompotato or of French fries, crisps or potato powder from potatoes. Thereis a particular interest in improving the starches in such a way, thatthey show a reduced “cold sweetening”, i.e. a decreased release ofreduced sugars (especially glucose) during long-term storage at lowtemperatures. Specifically potatoes are often stored at temperatures of4-8° C. in order to minimize the degradation of starch during storage.The reducing sugars released thereby, in particular glucose, lead toundesired browning reactions (so-called Maillard reactions) in theproduction of French fries and crisps.

Starch which can be isolated from plants is often adapted to certainindustrial purposes by means of chemical modifications which are usuallytime-consuming and expensive. Therefore it is desirable to findpossibilities to produce plants synthesizing a starch the properties ofwhich already meet the demands of the processing industry.

Conventional methods for producing such plants are classical breedingmethods and the production of mutants. Thus, for example, a mutant wasproduced from maize synthesizing starch with an altered viscosity (U.S.Pat. No. 5,331,108) and a maize variety (waxy maize) was established bymeans of breeding the starch of which consists of almost 100%amylopectin (Akasuka and Nelson, J. Biol. Chem. 241 (1966), 2280-2285).Furthermore, mutants of potato and pea have been described whichsynthesize starches with a high amylose content (70% in maize or up to50% in pea). These mutants have so far not been characterized on themolecular level and therefore do not allow for the production ofcorresponding mutants in other starch-storing plants.

Alternatively, plants synthesizing starch with altered properties may beproduced by means of recombinant DNA techniques. In various cases, forexample, the recombinant modification of potato plants aiming ataltering the starch synthesized in these plants has been described (e.g.WO 92/11376; WO 92/14827). However, in order to make use of recombinantDNA techniques, DNA sequences are required the gene products of whichinfluence starch synthesis, starch modification or starch degradation.

Therefore, the problem underlying the present invention is to providenucleic acid molecules and methods which allow for the alteration ofplants in such a way, that they synthesize a starch which differs fromstarch naturally synthesized in plants with respect to its physicaland/or chemical properties, in particular a highly amylose-containingstarch, and is therefore more suitable for general and/or particularuses.

DETAILED DESCRIPTION OF THE INVENTION

This problem is solved by the provision of the embodiments described inthe claims.

Therefore, the present invention relates to nucleic acid moleculesencoding a protein with the amino acid sequence indicated in Seq ID No.2. Such proteins are present in the plastids of plant cells, bound tostarch granules as well as in free, i.e. soluble form. During theexpression of E. coli, the enzyme activity of such proteins leads to anincreased phosphorylation of the glycogen synthesized within the cells.The molecular weight of these proteins lies within the range of 140-160kD if it is assessed by means of a SDS gel electrophoresis.

The present invention further relates to nucleic acid moleculescomprising a sequence with the nucleotide sequence indicated in Seq IDNo. 1, particularly the coding region indicated in Seq ID No. 1.

Nucleic acid molecules encoding a protein from potato, which in theplastids of the cells is partly granule-bound, and hybridizing to theabove-mentioned nucleic acid molecules of the invention or theircomplementary strand are also the subject matter of the presentinvention. In this context the term “hybridization” signifieshybridization under conventional hybridizing conditions, preferablyunder stringent conditions as described for example in Sambrook et al.,Molecular Cloning, A Laboratory Manual, 2^(nd) Edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). These nucleicacid molecules hybridizing with the nucleic acid molecules of theinvention may principally be derived from any desired organism (i.e.prokaryotes or eukaryotes, in particular from bacteria, fungi, alga,plants or animal organisms) comprising such nucleic acid molecules. Theyare preferably derived from monocotyledonous or dicotyledonous plants,particularly from useful plants, and particularly preferred fromstarch-storing plants.

Nucleic acid molecules hybridizing to the molecules according to theinvention may be isolated e.g. from genomic or from cDNA libraries ofvarious organisms.

Thereby, the identification and isolation of such nucleic acid moleculesmay take place by using the molecules according to the invention orparts of these molecules or, as the case may be, the reverse complementstrands of these molecules, e.g. by hybridization according to standardmethods (see e.g. Sambrook et al., 1989, Molecular Cloning, A LaboratoryManual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.).

As a probe for hybridization e.g. nucleic acid molecules may be usedwhich exactly or basically contain the nucleotide sequence indicatedunder Seq ID No. 1 or parts thereof. The DNA fragments used ashybridization probe may also be synthetic DNA fragments which wereproduced by means of the conventional DNA synthesizing methods and thesequence of which is basically identical with that of a nucleic acidmolecule of the invention. After identifying and isolating geneshybridizing to the nucleic acid sequences according to the invention,the sequence has to be determined and the properties of the proteinsencoded by this sequence have to be analyzed.

Furthermore, the present invention relates to nucleic acid molecules thesequences of which, compared to the sequences of the above-mentionedmolecules, are degenerated due to the genetic code and which encode aprotein which in the plastids of plant cells is partly granule-bound.

Fragments, derivatives and allelic variants of the above-mentionednucleic acid molecules, which encode the above-mentioned protein arealso the subject matter of the present invention. Thereby, fragments aredescribed as parts of the nucleic acid molecules which are long enoughin order to encode the above-described protein. In this context, theterm derivative signifies that the sequences of these molecules differfrom the sequences of the above-mentioned nucleic acid molecules at oneor more positions and exhibit a high degree of homology to the sequencesof these molecules. Hereby, homology means a sequence identity of atleast 40%, in particular an identity of at least 60%, preferably of morethan 80% and still more preferably a sequence identity of more than 90%.The deviations occurring when comparing with the above-described nucleicacid molecules might have been caused by deletion, substitution,insertion or recombination.

Moreover, homology means that functional and/or structural equivalenceexists between the respective nucleic acid molecules or the proteinsthey encode. The nucleic acid molecules, which are homologous to theabove-described nucleic acid molecules and represent derivatives ofthese molecules, are generally variations of these nucleic acidmolecules, that constitute modifications which exert the same biologicalfunction. These variations may be naturally occurring variations, forexample sequences from different organisms, or mutations, whereby thesemutations may have occurred naturally or they may have been introduceddeliberately. Moreover the variations may be synthetically producedsequences.

The allelic variants may be naturally occurring as well as syntheticallyproduced variants or variants produced by recombinant DNA techniques.

The proteins encoded by the various variants of the nucleic acidmolecules according to the invention exhibit certain commoncharacteristics. Enzyme activity, molecular weight, immunologicreactivity, conformation etc. may belong to these characteristics aswell as physical properties such as the mobility in gel electrophoresis,chromatographic characteristics, sedimentation coefficients, solubility,spectroscopic properties, stability, pH-optimum, temperature-optimumetc.

The nucleic acid molecules of the invention may principally be derivedfrom any organism expressing the described proteins. They are preferablyderived from plants, in particular from starch-synthesizing orstarch-storing plants. Cereals (such as barley, rye, oats, wheat etc.),maize, rice, pea, cassava, potato etc. are particularly preferred. Theycan also be produced by means of synthesis methods known to the skilledperson.

The nucleic acid molecules of the invention may be DNA molecules, suchas cDNA or genomic DNA, as well as RNA molecules.

Furthermore, the invention relates to vectors, especially plasmids,cosmids, viruses, bacteriophages and other vectors common in geneticengineering, which contain the above-mentioned nucleic acid molecules ofthe invention.

In a preferred embodiment the nucleic acid molecules contained in thevectors are linked to regulatory elements that ensure the transcriptionand synthesis of a translatable RNA in prokaryotic and eukaryotic cells.

In a further embodiment the invention relates to host cells, inparticular prokaryotic or eukaryotic cells, which have been transformedand/or recombinantly manipulated by an above-mentioned nucleic acidmolecule of the invention or by a vector of the invention, as well ascells derived from such cells and containing a nucleic acid molecule ofthe invention or a vector of the invention. This is preferably abacterial cell or a plant cell.

It was now found that the protein encoded by the nucleic acid moleculesof the invention influences the starch synthesis or modification andthat changes in the amount of the protein in plant cells lead to changesin the starch metabolism of the plant, especially to the synthesis ofstarch with modified physical and chemical properties.

By providing the nucleic acid molecules of the invention it is possibleto produce plants by means of recombinant DNA techniques synthesizing amodified starch which differs from the starch synthesized in wildtypeplants with respect to its structure and its physical and chemicalproperties. For this purpose, the nucleic acid molecules of theinvention are linked to regulatory elements, which ensure thetranscription and translation in plant cells, and they are introducedinto the plant cells.

Therefore, the present invention also relates to transgenic plant cellscontaining a nucleic acid molecule of the invention whereby the same islinked to regulatory elements which ensure the transcription in plantcells. The regulatory elements are preferably heterologous with respectto the nucleic acid molecule.

By means of methods known to the skilled person the transgenic plantcells can be regenerated to whole plants. The plants obtainable byregenerating the transgenic plant cells of the invention are also thesubject-matter of the present invention. A further subject-matter of theinvention are plants which contain the above-described transgenic plantcells. The transgenic plants may in principle be plants of any desiredspecies, i.e. they may be monocotyledonous as well as dicotyledonousplants. These are preferably useful plants, in particular starch-storingplants such as cereals (rye, barley, oats, wheat etc.), rice, maize,peas, cassava and potatoes.

Due to the expression or the additional expression of a nucleic acidmolecule of the invention, the transgenic plant cells and plants of theinvention synthesize a starch which is modified when compared to starchfrom wildtype-plants, i.e. non-transformed plants, particularly withrespect to the viscosity of aqueous solutions of this starch and/or tothe phosphate content. The latter is generally increased in the starchof transgenic plant cells or plants, this altering the physicalproperties of the starch.

Therefore, the starch obtainable from the transgenic plant cells andplants of the invention is also the subject-matter of the presentinvention.

A further subject-matter of the present invention is a method for theproduction of a protein which is present in plant cells in granule-boundform as well as in soluble from, in which host cells of the inventionare cultivated under conditions that allow for the expression of theprotein and in which the protein is then isolated from the cultivatedcells and/or the culture medium.

Furthermore, the invention relates to proteins encoded by the nucleicacid molecules of the invention as well as to proteins obtainable by theabove-described method. These are preferably proteins encoded by nucleargenes and which are localized in the plastids. In the plastids theseenzymes are present in granule-bound as well as in free form. In an SDSgel electrophoresis, the respective proteins from Solanum tuberosumexhibit a molecular weight of 140-160 kD and, during the expression ofE. coli, lead to an increased phosphorylation of the glycogensynthesized within the cells.

A further subject-matter of the invention are antibodies whichspecifically recognize a protein of the invention. These may bemonoclonal as well as polyclonal antibodies.

Furthermore, the present invention relates to nucleic acid moleculesspecifically hybridizing with a nucleic acid molecule of the inventionand exhibiting a length of at least 15 nucleotides. In this contextspecifically hybridizing signifies that under conventional hybridizationconditions, preferably under stringent conditions, cross-hybridizationwith sequences encoding other proteins does not significantly occur.Such nucleic acid molecules preferably have a length of at least 20,more preferably a length of at least 50 and most preferably a length ofat least 100 nucleotides. Such molecules can be used, for example, asPCR primers, as hybridization probes or as DNA molecules which encodeantisense RNA.

Furthermore, it was found that it is possible to influence theproperties of the starch synthesized in plant cells by reducing theamount of proteins encoded by the nucleic acid molecules according tothe invention in the cells. This reduction may be effected, for example,by means of antisense expression of the nucleic acid molecules of theinvention, expression of suitable ribozymes or cosuppression.

Therefore, DNA molecules encoding an antisense RNA which iscomplementary to transcripts of a DNA molecule of the invention are alsothe subject-matter of the present invention, as well as these antisensemolecules. Thereby, complementary does not signify that the encoded RNAhas to be 100% complementary. A low degree of complementarity issufficient, as long as it is high enough in order to inhibit theexpression of a protein of the invention upon expression in plant cells.The transcribed RNA is preferably at least 90% and most preferably atleast 95% complementary to the transcript of the nucleic acid moleculeof the invention. In order to cause an antisense-effect during thetranscription in plant cells such DNA molecules have a length of atleast 15 bp, preferably a length of more than 100 bp and most preferablya length of more than 500 bp, however, usually less than 5000 bp,preferably shorter than 2500 bp.

The invention further relates to DNA molecules which, during expressionin plant cells, lead to the synthesis of an RNA which in the plant cellsdue to a cosupression-effect reduces the expression of the nucleic acidmolecules of the invention encoding the described protein. The principleof the cosupression as well as the production of corresponding DNAsequences is precisely described, for example, in WO 90/12084. Such DNAmolecules preferably encode a RNA having a high degree of homology totranscripts of the nucleic acid molecules of the invention. It is,however, not absolutely necessary that the coding RNA is translatableinto a protein.

In a further embodiment the present invention relates to DNA moleculesencoding an RNA molecule with ribozyme activity which specificallycleaves transcripts of a DNA molecule of the invention as well as theseencoded RNA molecules.

Ribozymes are catalytically active RNA molecules capable of cleaving RNAmolecules and specific target sequences. By means of recombinant DNAtechniques it is possible to alter the specificity of ribozymes. Thereare various classes of ribozymes. For practical applications aiming atthe specific cleavage of the transcript of a certain gene, use ispreferably made of representatives of two different groups of ribozymes.The first group is made up of ribozymes which belong to the group Iintron ribozyme type. The second group consists of ribozymes which as acharacteristic structural feature exhibit the so-called “hammerhead”motif. The specific recognition of the target RNA molecule may bemodified by altering the sequences flanking this motif. By base pairingwith sequences in the target molecule these sequences determine theposition at which the catalytic reaction and therefore the cleavage ofthe target molecule takes place. Since the sequence requirements for anefficient cleavage are extremely low, it is in principle possible todevelop specific ribozymes for practically each desired RNA molecule.

In order to produce DNA molecules encoding a ribozyme which specificallycleaves transcripts of a DNA molecule of the invention, for example aDNA sequence encoding a catalytic domain of a ribozyme is bilaterallylinked with DNA sequences which are homologous to sequences of thetarget enzyme.

Sequences encoding the catalytic domain may for example be the catalyticdomain of the satellite DNA of the SCMo virus (Davies et al., Virology177 (1990), 216-224) or that of the satellite DNA of the TobR virus(Steinecke et al., EMBO J. 11 (1992), 1525-1530; Haseloff and Gerlach,Nature 334 (1988), 585-591). The DNA sequences flanking the catalyticdomain are preferably derived from the above-described DNA molecules ofthe invention.

In a further embodiment the present invention relates to vectorscontaining the above-described DNA molecules, in particular those inwhich the described DNA molecules are linked with regulatory elementsensuring the transcription in plant cells.

Furthermore, the present invention relates to host cells containing thedescribed DNA molecules or vectors. The host cell may be a prokaryoticcell, such as a bacterial cell, or a eukaryotic cell. The eucaryotichost cells are preferably plant cells.

Furthermore, the invention relates to transgenic plant cells containingan above-described DNA molecule encoding an antisense-RNA, a ribozyme oran RNA which leads to a cosuppression effect, whereby the DNA moleculeis linked to DNA elements ensuring the transcription in plant cells.These transgenic plant cells may be regenerated to whole plantsaccording to well-known techniques. Thus, the invention also relates toplants which may be obtained through regeneration from the describedtransgenic plant cells, as well as to plants containing the describedtransgenic plant cells. The transgenic plants themselves may be plantsof any desired plant species, preferably useful plants, particularlystarch-storing ones, as indicated above.

Due to the expression of the described DNA molecules encoding antisenseRNA, a ribozyme or a cosupression RNA in the transgenic plant cells theamount of proteins encoded by the DNA molecules of the invention whichare present in the cells in endogenic form, is reduced. Surprisingly,this reduction leads to a drastic change of the physical and chemicalproperties of the starch synthesized in the plant cells, in particularwith respect to the viscous properties of the aqueous solutions of thisstarch, to the phosphate content as well as to the release of reducingsugars in the storage of the plant cells or plant parts at lowtemperatures. The properties of the starch synthesized in the transgenicplant cells is explicitely described below.

Thus, the starch obtainable from the described transgenic plant cellsand plants is also the subject matter of the present invention.

Furthermore, the invention relates to the antisense RNA moleculesencoded by the described DNA molecules, as well as to RNA molecules withribozyme activity and RNA molecules which lead to a cosupression effectwhich are obtainable, for example, by means of transcription.

A further subject-matter of the invention is a method for the productionof transgenic plant cells, which in comparison to non-transformed cellssynthesize a modified starch. In this method the amount of proteinsencoded by the DNA molecules of the invention, which are present in thecells in endogenic form, is reduced in the plant cells.

In a preferred embodiment this reduction is effected by means of anantisense effect. For this purpose the DNA molecules of the invention orparts thereof are linked in antisense orientation with a promoterensuring the transcription in plant cells and possibly with atermination signal ensuring the termination of the transcription as wellas the polyadenylation of the transcript. In order to ensure anefficient antisense effect in the plant cells the synthesized antisenseRNA should exhibit a minimum length of 15 nucleotides, preferably of atleast 100 nucleotides and most preferably of more than 500 nucleotides.Furthermore, the DNA sequence encoding the antisense RNA should behomologous with respect to the plant species to be transformed. However,DNA sequences exhibiting a high degree of homology to DNA sequenceswhich are present in the cells in endogenic form may also be used,preferably with an homology of more than 90% and most preferably with anhomology of more than 95%.

In a further embodiment the reduction of the amount of proteins encodedby the DNA molecules of the invention is effected by a ribozyme effect.The basic effect of ribozymes as well as the construction of DNAmolecules encoding such RNA molecules have already been described above.In order to express an RNA with ribozyme activity in transgenic cellsthe above described DNA molecules encoding a ribozyme are linked withDNA elements which ensure the transcription in plant cells, particularlywith a promoter and a termination signal. The ribozymes synthesized inthe plant cells lead to the cleavage of transcripts of DNA molecules ofthe invention which are present in the plant cells in endogenic form.

A further possibility in order to reduce the amount of proteins encodedby the nucleic acid molecules of the invention is cosupression.Therefore, the plant cells obtainable by the method of the invention area further subject matter. These plant cells are characterized in thattheir amount of proteins encoded by the DNA molecules of the inventionis reduced and that in comparison to wildtype cells they synthesize amodified starch.

Furthermore, the invention relates to plants obtainable by regenerationof the described plant cells as well as to plants containing thedescribed cells of the invention.

The starch obtainable from the described plant cells and plants is alsothe subject-matter of the present invention. This starch differs fromstarch obtained from non-transformed cells or plants in its physicaland/or chemical properties. When compared to starch from wildtypeplants, the starch exhibits a reduced phosphate content. Moreover, theaqueous solutions of this starch exhibit modified viscous properties.

In a preferred embodiment the phosphate content of the described starchis reduced by at least 50%, more preferably by at least 75% and in aparticularly preferred embodiment by more than 80% in comparison tostarch derived from wildtype plants.

The modified viscosity of the aqueous solution of this starch is itsmost advantageous feature.

A well-established test for determining the viscosity is the so-calledBrabender test. This test is carried out by using an appliance which isfor example known as viscograph E. This equipment is produced and sold,among others, by Brabender fOHG Duisburg (Germany).

The test basically consists in first heating starch in the presence ofwater in order to assess when hydratization and the swelling of thestarch granules takes place. This process which is also namedgelatinization or pastification is based on the dissolving the hydrogenbonds and involves a measurable increase of the viscosity in the starchsuspension. While further heating after gelatinization leads to thecomplete dissolving of the starch particles and to a decrease ofviscosity, the immediate cooling after gelatinization typically leads toa increase in the viscosity (see FIG. 3). The result of the Brabendertest is a graph which shows the viscosity depending on time, whereby atfirst the solution is heated to above the gelatinization temperature andthen cooled.

The analysis of the Brabender graph is generally directed to determiningthe pastification temperature, the maximum viscosity during heating, theincrease in viscosity during cooling, as well as the viscosity aftercooling. These parameters are important characteristics when it comes tothe quality of a starch and the possibilty to use it for variouspurposes.

The starch which may for example be isolated from potato plants in whichthe amount of proteins of the invention within the cells was reduced bymeans of an antisense effect, showed characteristics strongly deviatingfrom the characteristics of starch isolated from wildtype plants.Compared with these it only shows a low increase in viscosity duringheating, a low maximum viscosity as well as a stronger increase inviscosity during cooling (see FIGS. 3, 4 and 5).

In a preferred embodiment the invention relates to starch, the aqueoussolutions of which exhibit the characteristic viscous propertiesdepicted in FIGS. 4 or 5. Particularly under the conditions mentioned inExample 8 a for determining the viscosity with the help of a Brabenderviscosimeter, the modified starch, when compared to wildtype plants,exhibits the characteristic of only a low increase in viscosity whenheating the solution. This offers the opportunity of using the starchfor the production of highly-concentrated glues.

Moreover, after reaching maximum viscosity, there is only a low decreasein viscosity in the case of the modified starch. On the other hand theviscosity increases strongly on cooling; thus, the viscosity of modifiedstarch is higher than the viscosity of starch from wildtype plants.

By reducing the amount of proteins of the invention in transgenic plantcells it is furthermore possible to produce a starch which has theeffect that when plant parts containing this starch are stored at lowtemperatures, in particular at 4-8° C., less reducing sugars arereleased than is the case which starch from non-transformed cells. Thisproperty is particularly advantageous, for example, for providingpotatoes which during storage at low temperatures release less reducingsugars and thus exhibit a reduced cold sweetening. Such potatoes areparticularly suitable for producing French fries, crisps or similarproducts since undesirable browning-reactions (Maillard reactions) areavoided or at least strongly reduced during use.

In a particularly preferred embodiment of the present invention not onlythe synthesis of a protein of the invention is reduced in thetransformed plant cells, but moreover also the synthesis of at least onefurther enzyme involved in starch synthesis and/or modification. In thiscontext, for example, starch granule-bound starch synthases or branchingenzymes are preferred. Surprisingly, it was found that potato plants inwhich the synthesis of the proteins of the invention as well as of thebranching enzyme is reduced due to an antisense effect synthesize astarch which in its properties strongly deviates from starch of wildtypeplants.

When compared to wildtype starch, the aqueous solutions of this modifiedstarch show almost no increase in viscosity during heating or cooling(cf. FIG. 6).

Furthermore, a microscopical analysis of the starch granules before andafter heating clearly shows that, when compared to wildtype plants, thestarch granules of plants modified in such a way are not open but remainbasically unchanged in their structure. Thus, this is a starch which isresistent to the heating process. If the amylose content of this starchis determined by means of the method described in the Examples, amylosecontents of more than 50%, preferably of more than 60% and mostpreferably of more than 70% are measured for this starch. The aqueoussolutions of the starch isolated from this plants preferably show thecharacteristic viscous properties depicted in FIG. 6.

Such a highly amylose-containing starch of the invention offers a numberof advantages for various uses when compared to wildtype plants. Thus,highly amylose-containing starches have a high potential for the use infoils and films. The foils and films produced on the basis of highlyamylose-containing starches, which may be used in wide areas of thepackaging industry, have the essential advantage of being biodegradable.Apart from this use which is basically covered by classical,petrochemically produced polymers, amylose has further unique fields ofapplication which are caused by the amylose's property to form helices.The helix formed by the amylose is internally hydrophobic and externallyhydrophilic. Due to this, amylose may be used for the complexation andmolecular encapsulation of low molecular or also of high molecularsubstances. Examples therefore are:

the molecular encapsulation of vitamins and substances for theprotection against oxidation, volatilization, thermal degradation or thetransition into an aqueous environment;

the molecular encapsulation of aromatic substances for increasing thesolubility;

the molecular encapsulation of fertilizers/pesticides for stabilizationand controlled release;

the molecular encapsulation of medical substances for stabilizing thedosage-control and for the controlled release of retarding preparations.

Another important property of amylose is the fact that it is a chiralmolecule. Due to the chirality it may preferably be used afterimmobilization, e.g. on a column for separating enantiomers.

Furthermore, it was surprisingly found that starch which may be isolatedfrom potato plants in which the amount of proteins of the invention inthe cells was reduced due to an antisense effect, in combination with areduction of the proteins exhibiting the enzymatic activity of a starchgranule-bound starch synthase of the isotype I (GBSSI) exhibitscharacteristics which strongly deviate from the characteristics ofstarch which may be isolated from wildtype plants. When compared tostarch from wildtype plants, the aqueous solutions of this starch onlyshow a low increase in viscosity during heating, a low maximum viscosityas well as almost no increase in viscosity during cooling (cf. FIG. 7).If the amylose/amylopectin ratio of this starch is determined, thisstarch is characterized in that almost no amylose can be measured. Theamylose content of this starch is preferably below 5% and mostpreferably below 2%. The starch of the invention furthermore differsfrom the known starch which may be produced in transgenic potato plantsby inhibiting the GBSSI gene solely by means of recombinant DNAtechniques. Thus, this starch shows a strong increase in viscosityduring heating. The aqueous solutions of the starch of the inventionpreferably show the characteristic viscous properties depicted in FIG.7. Particularly under the conditions for determining the viscosity bymeans of a Rapid Visco Analyser described in Example 13, the modifiedstarch has the characteristic of only exhibiting a low viscosityincrease during heating when compared to wildtype starch, but also whencompared to waxy starch. This offers the opportunity to use the starchof the invention for the production of highly-concentrated glues. Themodified starch furthermore has the property that there is only a lowdecrease of viscosity after reaching the maximum viscosity, as well asalmost no increase in viscosity during cooling.

Possibilities in order to reduce the activity of a branching enzyme inplant cells were already described, for example in WO 92/14827 and WO95/26407. The reduction of the activity of a starch granule-bound starchsynthase of the isotype I (GBSSI) may be carried out by using methodsknown to the skilled person, e.g. by means of an antisense effect. DNAsequences encoding a GBSSI from potatoe are for example known fromHegersberg (dissertation (1988) University of Cologne), Visser et al.(Plant Sci. 64 (1989), 185-192) or van der Leiy et al. (Mol. Gen. Genet.228 (1991), 240-248).

The method of the invention may in principle be used for any kind ofplant species. Monocotyledonous and dicotyledonous plants are ofinterest, in particular useful plants and preferably starch-storingplants such as cereals (rye, barley, oats, wheat etc.), rice, maize,pea, cassava and potatoes.

Within the framework of the present invention the term “regulatory DNAelements ensuring the transcription in plant cells” are DNA regionswhich allow for the initiation or the termination of transcription inplant cells. DNA regions ensuring the initiation of transcription are inparticular promoters.

For the expression of the various above-described DNA molecules of theinvention in plants any promoter functioning in plant cells may be used.The promoter may be homologous or heterologous with respect to the usedplant species. Use may, for example, be made of the 35S promoter of thecauliflower mosaic virus (Odell et al., Nature 313 (1985), 810-812)which ensures a constitutive expression in all plant tissues and also ofthe promoter construct described in WO/9401571. However, use may also bemade of promoters which lead to an expression of subsequent sequencesonly at a point of time determined by exogenous factors (such as inWO/9307279) or in a particular tissue of the plant (see e.g. Stockhauset al., EMBO J. 8 (1989), 2245-2251). Promoters which are active in thestarch-storing parts of the plant to be transformed are preferably used.In the case of potato these parts are the potato seeds, in the case ofpotatoes the tubers. In order to transform potatoes the tuber-specificB33-promoter (Rocha-Sosa et al., EMBO J. 8 (1989), 23-29) may be usedparticularly, but not exclusively.

Apart from promoters, DNA regions initiating transcription may alsocontain DNA sequences ensuring a further increase of transcription, suchas the so-called enhancer-elements. Furthermore, the term “regulatoryDNA elements” may also comprise termination signals which serve tocorrectly end the transcription and to add a poly-A-tail to thetranscript which is believed to stabilize the transcripts. Such elementsare described in the literature and can be exchanged as desired.Examples for such termination sequences are the 3′-nontranslatableregions comprising the polyadenylation signal of the nopaline synthasegene (NOS gene) or the octopine synthase gene (Gielen et al., EMBO J. 8(1989), 23-29) from agrobacteria, or the 3′-nontranslatable regions ofthe genes of the storage proteins from soy bean as well as the genes ofthe small subunit of ribulose-1,5-biphosphate-carboxylase (ssRUBISCO).

The introduction of the DNA molecules of the invention into plant cellsis preferably carried out using plasmids. Plasmids ensuring a stableintegration of the DNA into the plant genome are preferred.

In the examples of the present invention use is made of the binaryvector pBinAR (Höfgen and Willmitzer, Plant Sci. 66 (1990), 221-230).This vector is a derivative of the binary vector pBin19 (Bevan, Nucl.Acids Res. 12 (1984), 8711-8721), which may commercially be obtained(Clontech Laboratories, Inc. USA).

However, use may be made of any other plant transformation vector whichcan be inserted into a expression cassette and which ensures theintegration of the expression cassette into the plant genome.

In order to prepare the introduction of foreign genes in higher plants alarge number of cloning vectors are at disposal, containing areplication signal for E. coli and a marker gene for the selection oftransformed bacterial cells. Examples for such vectors are pBR322, pUCseries, M13mp series, pACYC184 etc. The desired sequence may beintegrated into the vector at a suitable restriction site. The obtainedplasmid is used for the transformation of E. coli cells. Transformed E.coli cells are cultivated in a suitable medium and subsequentlyharvested and lysed. The plasmid is recovered by means of standardmethods. As an analyzing method for the characterization of the obtainedplasmid DNA use is generally made of restriction analysis and sequenceanalysis. After each manipulation the plasmid DNA may be cleaved and theobtained DNA fragments may be linked to other DNA sequences.

In order to introduce DNA into plant host cells a wide range oftechniques are at disposal. These techniques comprise the transformationof plant cells with T-DNA by using Agrobacterium tumefaciens orAgrobacterium rhizogenes as transformation medium, the fusion ofprotoplasts, the injection and the electroporation of DNA, theintroduction of DNA by means of the biolistic method as well as furtherpossibilities.

In the case of injection and electroporation of DNA into plant cells,there are no special demands made to the plasmids used. Simple plasmidssuch as pUC derivatives may be used. However, in case that whole plantsare to be regenerated from cells transformed in such a way, a selectablemarker gene should be present.

Depending on the method of introducing desired genes into the plantcell, further DNA sequences may be necessary. If the Ti- or Ri-plasmidis used e.g. for the transformation of the plant cell, at least theright border, more frequently, however, the right and left border of theTi- and Ri-plasmid T-DNA has to be connected to the foreign gene to beintroduced as a flanking region.

If Agrobacteria are used for transformation, the DNA which is to beintroduced must be cloned into special plasmids, namely either into anintermediate vector or into a binary vector. Due to sequences homologousto the sequences within the T-DNA, the intermediate vectors may beintegrated into the Ti- or Ri-plasmid of the Agrobacterium due tohomologous recombination. This also contains the vir-region necessaryfor the transfer of the T-DNA. Intermediate vectors cannot replicate inAgrobacteria. By means of a helper plasmid the intermediate vector maybe transferred to Agrobacterium tumefaciens (conjugation). Binaryvectors may replicate in E. coli as well as in Agrobacteria. Theycontain a selectable marker gene as well as a linker or polylinker whichis framed by the right and the left T-DNA border region. They may betransformed directly into the Agrobacteria (Holsters et al. Mol. Gen.Genet. 163 (1978), 181-187). The plasmids used for the transformation ofthe Agrobacteria further comprise a selectable marker gene, such as theNPT II gene which allows for selecting transformed bacteria. TheAgrobacterium acting as host cell should contain a plasmid carrying avir-region. The vir-region is necessary for the transfer of the T-DNAinto the plant cell. Additional T-DNA may be present. The Agrobacteriumtransformed in such a way is used for the transformation of plant cells.

The use of T-DNA for the transformation of plant cells was investigatedintensely and described sufficiently in EP 120 516; Hoekema, In: TheBinary Plant Vector System Offsetdrukkerij Kanters B. V., Alblasserdam(1985), Chapter V; Fraley et al., Crit. Rev. Plant. Sci., 4, 1-46 and Anet al. EMBO J. 4 (1985), 277-287. Some binary vectors may already beobtained commercially, such as pBIN19 (Clontech Laboratories, Inc.,USA).

For transferring the DNA into the plant cells, plant explants maysuitably be co-cultivated with Agrobacterium tumefaciens orAgrobacterium rhizogenes. From the infected plant material (e.g. piecesof leaves, stem segments, roots, but also protoplasts orsuspension-cultivated plant cells) whole plants may then be regeneratedin a suitable medium which may contain antibiotics or biozides for theselection of transformed cells. The plants obtained in such a way maythen be examined as to whether the introduced DNA is present or not.

Once the introduced DNA has been integrated in the genome of the plantcell, it usually continues to be stable there and also remains withinthe descendants of the originally transformed cell. It usually containsa selectable marker which confers resistance against biozides or againstan antibiotic such as kanamycin, G 418, bleomycin, hygromycin orphosphinotricine etc. to the transformed plant cells. The individuallyselected marker should therefore allow for a selection of transformedcells against cells lacking the introduced DNA.

The transformed cells grow in the usual way within the plant (see alsoMcCormick et al., Plant Cell Reports 5 (1986), 81-84). The resultingplants can be cultivated in the usual way and cross-bred with plantshaving the same transformed genetic heritage or another geneticheritage. The resulting hybrid individuals have the correspondingphenotypic properties.

Two or more generations should be grown in order to ensure whether thephenotypic feature is kept stably and whether it is transferred.Furthermore, seeds should be harvested in order to ensure that thecorresponding phenotype or other properties will remain.

Due to its properties the starch obtained from the plant cells or fromthe plants of the invention is not only suitable for the specificpurposes already mentioned herein, but also for various industrial uses.

Basically, starch can be subdivided into two major fields. One fieldcomprises the hydrolysis products of starch and the so-called nativestarches. The hydrolysis products essentially comprise glucose andglucans components obtained by enzymatic or chemical processes. They canbe used for further processes, such as fermentation and chemicalmodifications. In this context, it might be of importance that thehydrolysis process can be carried out simply and inexpensively.Currently, it is carried out substantially enzymatically usingamyloglucosidase. It is thinkable that costs might be reduced by usinglower amounts of enzymes for hydrolysis due to changes in the starchstructure, e.g. increasing the surface of the grain, improveddigestibility due to less branching or a steric structure, which limitsthe accessibility for the used enzymes.

The use of the so-called native starch which is used because of itspolymer structure can be subdivided into two further areas:

(a) Use in Foodstuffs

Starch is a classic additive for various foodstuffs, in which itessentially serves the purpose of binding aqueous additives and/orcauses an increased viscosity or an increased gel formation. Importantcharacteristic properties are flowing and sorption behavior, swellingand pastification temperature, viscosity and thickening performance,solubility of the starch, transparency and paste structure, heat, shearand acid resistance, tendency to retrogradation, capability of filmformation, resistance to freezing/thawing, digestibility as well as thecapability of complex formation with e.g. inorganic or organic ions.

(b) Use in Non-Foodstuffs

The other major field of application is the use of starch as an adjuvantin various production processes or as an additive in technical products.The major fields of application for the use of starch as an adjuvantare, first of all, the paper and cardboard industry. In this field, thestarch is mainly used for retention (holding back solids), for sizingfiller and fine particles, as solidifying substance and for dehydration.In addition, the advantageous properties of starch with regard tostiffness, hardness, sound, grip, gloss, smoothness, tear strength aswell as the surfaces are utilized.

Within the paper production process, a differentiation can be madebetween four fields of application, namely surface, coating, mass andspraying.

The requirements on starch with regard to surface treatment areessentially a high degree of brightness, corresponding viscosity, highviscosity stability, good film formation as well as low formation ofdust. When used in coating the solid content, a corresponding viscosity,a high capability to bind as well as a high pigment affinity play animportant role. As an additive to the mass rapid, uniform, loss-freedispersion, high mechanical stability and complete retention in thepaper pulp are of importance. When using the starch in spraying,corresponding content of solids, high viscosity as well as highcapability to bind are also significant.

A major field of application is, for instance, in the adhesive industry,where the fields of application are subdivided into four areas: the useas pure starch glue, the use in starch glues prepared with specialchemicals, the use of starch as an additive to synthetic resins andpolymer dispersions as well as the use of starches as extenders forsynthetic adhesives. 90% of all starch-based adhesives are used in theproduction of corrugated board, paper sacks and bags, compositematerials for paper and aluminum, boxes and wetting glue for envelopes,stamps, etc.

Another possible use as adjuvant and additive is in the production oftextiles and textile care products. Within the textile industry, adifferentiation can be made between the following four fields ofapplication: the use of starch as a sizing agent, i.e. as an adjuvantfor smoothing and strengthening the burring behavior for the protectionagainst tensile forces active in weaving as well as for the increase ofwear resistance during weaving, as an agent for textile improvementmainly after quality-deteriorating pretreatments, such as bleaching,dying, etc., as thickener in the production of dye pastes for theprevention of dye diffusion and as an additive for warping agents forsewing yarns.

Furthermore, starch may be used as an additive in building materials.One example is the production of gypsum plaster boards, in which thestarch mixed in the thin plaster pastifies with the water, diffuses atthe surface of the gypsum board and thus binds the cardboard to theboard. Other fields of application are admixing it to plaster andmineral fibers. In ready-mixed concrete, starch may be used for thedeceleration of the sizing process.

Furthermore, the starch is advantageous for the production of means forground stabilization used for the temporary protection of groundparticles against water in artificial earth shifting. According tostate-of-the-art knowledge, combination products consisting of starchand polymer emulsions can be considered to have the same erosion- andencrustation-reducing effect as the products used so far; however, theyare considerably less expensive.

Another field of application is the use of starch in plant protectivesfor the modification of the specific properties of these preparations.For instance, starches are used for improving the wetting of plantprotectives and fertilizers, for the dosed release of the activeingredients, for the conversion of liquid, volatile and/or odorousactive ingredients into microcristalline, stable, deformable substances,for mixing incompatible compositions and for the prolongation of theduration of the effect due to a reduced disintegration.

Starch may also be used in the fields of drugs, medicine and in thecosmetics industry. In the pharmaceutical industry, the starch may beused as a binder for tablets or for the dilution of the binder incapsules. Furthermore, starch is suitable as disintegrant for tabletssince, upon swallowing, it absorbs fluid and after a short time itswells so much that the active ingredient is released. For qualitativereasons, medicinal flowance and dusting powders are further fields ofapplication. In the field of cosmetics, the starch may for example beused as a carrier of powder additives, such as scents and salicylicacid. A relatively extensive field of application for the starch istoothpaste.

The use of starch as an additive in coal and briquettes is alsothinkable. By adding starch, coal can be quantitatively agglomeratedand/or briquetted in high quality, thus preventing prematuredisintegration of the briquettes. Barbecue coal contains between 4 and6% added starch, calorated coal between 0.1 and 0.5%. Furthermore, thestarch is suitable as a binding agent since adding it to coal andbriquette can considerably reduce the emission of toxic substances.

Furthermore, the starch may be used as a flocculant in the processing ofore and coal slurry.

Another field of application is the use as an additive to processmaterials in casting. For various casting processes cores produced fromsands mixed with binding agents are needed. Nowadays, the most commonlyused binding agent is bentonite mixed with modified starches, mostlyswelling starches.

The purpose of adding starch is increased flow resistance as well asimproved binding strength. Moreover, swelling starches may fulfill moreprerequisites for the production process, such as dispersability in coldwater, rehydratisability, good mixability in sand and high capability ofbinding water.

In the rubber industry starch may be used for improving the technicaland optical quality. Reasons for this are improved surface gloss, gripand appearance. For this purpose, the starch is dispersed on the stickyrubberized surfaces of rubber substances before the cold vulcanization.It may also be used for improving the printability of rubber.

Another field of application for the modified starch is the productionof leather substitutes.

In the plastics market the following fields of application are emerging:the integration of products derived from starch into the processingprocess (starch is only a filler, there is no direct bond betweensynthetic polymer and starch) or, alternatively, the integration ofproducts derived from starch into the production of polymers (starch andpolymer form a stable bond).

The use of the starch as a pure filler cannot compete with othersubstances such as talcum. This situation is different when the specificstarch properties become effective and the property profile of the endproducts is thus clearly changed. One example is the use of starchproducts in the processing of thermoplastic materials, such aspolyethylene. Thereby, starch and the synthetic polymer are combined ina ratio of 1:1 by means of coexpression to form a ‘master batch’, fromwhich various products are produced by means of common techniques usinggranulated polyethylene. The integration of starch in polyethylene filmsmay cause an increased substance permeability in hollow bodies, improvedwater vapor permeability, improved antistatic behavior, improvedanti-block behavior as well as improved printability with aqueous dyes.

Another possibility is the use of the starch in polyurethane foams. Dueto the adaptation of starch derivatives as well as due to theoptimization of processing techniques, it is possible to specificallycontrol the reaction between synthetic polymers and the starch's hydroxygroups. The results are polyurethane films having the following propertyprofiles due to the use of starch: a reduced coefficient of thermalexpansion, decreased shrinking behavior, improved pressure/tensionbehavior, increased water vapor permeability without a change in wateracceptance, reduced flammability and cracking density, no drop off ofcombustible parts, no halides and reduced aging. Disadvantages thatpresently still exist are reduced pressure and impact strength.

Product development of film is not the only option. Also solid plasticsproducts, such as pots, plates and bowls can be produced by means of astarch content of more than 50%. Furthermore, the starch/polymermixtures offer the advantage that they are much easier biodegradable.

Furthermore, due to their extreme capability to bind water, starch graftpolymers have gained utmost importance. These are products having abackbone of starch and a side lattice of a synthetic monomer grafted onaccording to the principle of radical chain mechanism. The starch graftpolymers available nowadays are characterized by an improved binding andretaining capability of up to 1000 g water per g starch at a highviscosity. These super absorbers are used mainly in the hygiene field,e.g. in products such as diapers and sheets, as well as in theagricultural sector, e.g. in seed pellets.

What is decisive for the use of the new starch modified by recombinantDNA techniques are, on the one hand, structure, water content, proteincontent, lipid content, fiber content, ashes/phosphate content,amylose/amylopectin ratio, distribution of the relative molar mass,degree of branching, granule size and shape as well as crystallization,and on the other hand, the properties resulting in the followingfeatures: flow and sorption behavior, pastification temperature,viscosity, thickening performance, solubility, paste structure,transparency, heat, shear and acid resistance, tendency toretrogradation, capability of gel formation, resistance tofreezing/thawing, capability of complex formation, iodine binding, filmformation, adhesive strength, enzyme stability, digestibility andreactivity. The most remarkable feature is viscosity.

Moreover, the modified starch obtained from the plant cells of theinvention may be subjected to further chemical modification, which willresult in further improvement of the quality for certain of theabove-described fields of application. These chemical modifications areprincipally known to the person skilled in the art. These areparticularly modifications by means of

acid treatment

oxidation and

esterification (formation of phosphate, nitrate, sulphate, xanthate,acetate and citrate starches. Further organic acids may also be used foresterification.)

formation of starch ethers (starch alkyl ether, O-allyl ether,hydroxylalkyl ether, O-carboxylmethyl ether, N-containing starch ethers,S-containing starch ethers)

formation of branched starches

formation of starch graft polymers.

The invention also relates to propagation material of the plants of theinvention, such as seeds, fruits, cuttings, tubers or root stocks,wherein this propagation material contains plant cells of the invention.

Deposits

The plasmids produced and/or used within the framework of the presentinvention have been deposited at the internationally acknowledgeddeposit “Deutsche Sammlung von Mikroorganismen (DSM)” in Braunschweig,Federal Republic of Germany, according to the requirements of theBudapest treaty for international acknowledgment of microorganismdeposits for patenting (deposit number; deposition date):

plasmid pBinAR Hyg (DSM 9505) (10/20/94) plasmid p33-anti-BE (DSM 6146)(08/20/90) plasmid pRL2 (DSM 10225) (09/04/95)

Media and Solutions Elution buffer: 25 mM Tris pH 8, 3 250 mM glycineDialysis buffer: 50 mM Tris-HCl pH 7, 0 50 mM NaCl 2 mM EDTA 14, 7 mMβ-mercaptoethanol 0, 5 mM PMSF Protein buffer: 50 mM sodium phosphatebuffer pH 7, 2 10 mM EDTA 0, 5 mM PMSF 14, 7 mM B-mercaptoethanol Lugolsolution: 12 g KI 6 g I₂ ad 1, 8 1 with ddH₂O 20 × SSC: 175.3 g NaCl88.2 g sodium citrate ad 1000 ml with ddH₂O ph 7, 0 with 10 N NaOH 10 xMEN: 200 mM MOPS 50 mM sodium acetate 10 mM EDTA pH 7, 0 NSEB buffer: 0,25 M sodium phosphate buffer pH 7, 2 7% SDS 1 mM EDTA 1% BSA (w/v)

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the plasmid p35S-anti-RL.

Plasmid Structure:

A=fragment A: CaMV 35S promoter, nt 6909-7437 (Franck et al., Cell 21(1980), 285-294)

B=fragment B: Asp718 fragment from pRL1 with a length of approximately1949 bp

Orientation relative to the promoter: anti-sense

The arrow indicates the direction of the open reading frame.

C=fragment C: nt 11748-11939 of the T-DNA of Ti-plasmid pTiACH5 T-DNA(Gielen et al., EMBO J. 3 (1984), 835-846)

FIG. 2 shows the plasmid pB33-anti-RL

Plasmid Structure:

A=fragment A: B33 promoter of the patatin gene B33 from Solanumtuberosum (Rocha-Sosa et al., EMBO J. 8 (1989), 23-29)

B=fragment B: Asp718 fragment from pRL1 with a length of approximately1949 bp

Orientation relative to the promoter: anti-sense

The arrow indicates the direction of the open reading frame.

C=fragment C: nt 11748-11939 of the T-DNA of Ti-plasmid pTiACH5 T-DNA(Gielen et al., EMBO J. 3 (1984), 835-846)

FIG. 3 shows a Brabender curve of a aqueous starch solution, recordedwith a Viskograph-E-type Brabender viscograph, which was isolated fromnon-transformed potato plants of the variety Désirée (see also Example8).

Thereby signifying: Drehm. torque [BE] Brabender unit Temp. temperatureA start of pastification B maximum degree of viscosity C start of the96° C. period D start of the cooling-off period E end of the cooling-offperiod F end of the end-50° C. period

The blue line indicates the viscosity; the red line stands fortemperature.

FIG. 4 shows a Brabender curve of a aqueous starch solution, recordedwith a Viskograph-E-type Brabender viscograph, which was isolated frompotato plants transformed with the plasmid p35S-anti-RL (see alsoExample 8). For the meaning of the abbreviations see FIG. 3.

FIG. 5 shows a Brabender curve of a aqueous solution of starch frompotatoes transformed with the plasmid pB33-anti-RL (see also Example 8),recorded with a Viskograph-E-type Brabender viscograph. For the meaningof the abbreviations see FIG. 3.

FIG. 6 shows curves of aqueous solutions of starch isolated from potatoplants (see also Example 12), which were recorded with a Rapid ViscoAnalyser. The red line stands for the temperature; the blue lines 1, 2,3 and 4 show the viscosities of the following starch solutions:

Line 1: starch isolated from wildtype plants,

Line 2: starch isolated from plants in which only the branching enzymewas inhibited (cf. Example 1 of patent application WO92/14827),

Line 3: starch isolated from plants in which merely the concentration ofthe proteins of the invention had been reduced (cf. Example 6).

Line 4: starch isolated from plants which had been transformed with theplasmid p35S-anti-RL in combination with the p35SH-anti-BE plasmid (cf.Example 12).

FIG. 7 shows curves of aqueous solutions of starch isolated from potatoplants (see also Example 13), which were recorded with a Rapid ViscoAnalyser. The red line stands for the temperature; the blue lines 1, 2,3 and 4 show the viscosities of the following starch solutions:

Line 1: starch isolated from wildtype plants,

Line 2: starch isolated from plants which had solely been transformedwith the plasmid pB33-anti-GBSSI (so-called waxy-potato),

Line 3: starch isolated from plants which had been solely transformedwith the plasmid p35S-anti-RL (cf. Example 6).

Line 4: starch isolated from plants which had been transformed with theplasmid pB33-anti-RL in combination with the plasmid pB33-anti-GBSSI(cf. Example 13).

EXPERIMENTAL METHODS AND EXAMPLES

The Examples illustrate the invention.

1. Cloning

For cloning in E. coli the vector pBluescriptSK was used.

For plant transformation the gene constructs were cloned into the binaryvector pBinAR (Höfgen and Willmitzer, Plant Sci. 66 (1990), 221-230) andB33-Hyg.

2. Bacterial Strains

For the Bluescript vector and for the pBinAR and B33-Hyg constructs usewas made of the E. coli strain DH5α (Bethesda Research Laboratories,Gaithersburgh, USA).

The transformation of plasmid in potato plants was carried out by meansof the Agrobacterium tumefaciens strain C58C1 pGV2260 (Deblaere et al.,Nucl. Acids Res. 13 (1985), 4777:4788).

3. Transformation of Agrobacterium Tumefaciens

The DNA transfer was carried out by means of direct transformationaccording to the method of Höfgen & Willmitzer (Nucleic Acids Res. 16(1988), 9877). The plasmid DNA of transformed Agrobacteria was isolatedaccording to the method of Birnboim & Doly (Nucleic Acids Res. 7 (1979),1513-1523) and electrophoretically analyzed after suitable restrictioncleavage.

4. Transformation of Potatoes

Ten small leaves of a sterile potato culture (Solanum tuberosum L. cv.Dësirëe) injured by a scalpel were treated with 10 ml MS medium(Murashige & Skoog, Physiol. Plant. 15 (1962), 473-497) with 2% sucrose.The medium contained 50 μl of a Agrobacterium tumefaciensovernight-culture grown under selection. After slightly shaking it for3-5 minutes, another incubation took place in darkness for two days. Theleaves were subsequently put on MS medium with 1,6% glucose, 5 mg/lnaphthyl acetic acid, 0,2 mg/l benzylaminopurine, 250 mg/l claforan, 50mg/l kanamycin or 1 mg/l hygromycin B, and 0,80% Bacto Agar for callusinduction. After a one-week incubation at 25° C. and 3000 lux the leaveswere put on MS-medium with 1,6% glucose, 1,4 mg/l zeatine ribose, 20mg/l naphthyl acetic acid, 20 mg/l giberellic acid, 250 mg/l claforan,50 mg/l kanamycin or 3 mg/l hygromycin B and 0,80% Bacto Agar for shootinduction.

5. Radioactive Marking of DNA Fragments

The radioactive marking of DNA fragments was carried out by means of aDNA-Random Primer Labeling Kits by Boehringer (Germany) according to themanufacturer's instructions.

6. Northern Blot Analysis

RNA was isolated from leave tissue according to standard protocols. 50μg of the RNA was separated on an agarose gel (1.5% agarose, 1×MENbuffer, 16.6% formaldehyde). After the gel run the gel was brieflywashed in water. The RNA was transferred to a Hybond N type nylonmembrane (Amersham, UK) with 20×SSC by means of capillary blot. Themembrane was subsequently baked in vacuum for two hours at 80° C.

The membrane was prehybridized in NSEB buffer for two hours at 68° C.and subsequently hybridized overnight in NSEB buffer in the presence ofthe radioactively marked probe at 68° C.

7. Plant Maintenance

Potato plants were kept in the greenhouse under the followingconditions:

light period 16 hours at 25000 lux and 22° C.

dark period 8 hours at 15° C.

atmospheric humidity 60%

8. Determination of the Amylose/Amylopectin Ratio in Starch Obtainedfrom Potato Plants

Starch was isolated from potato plants according to standard methods andthe amylose/amylopectin ratio was determined according to the methoddescribed by Hovenkamp-Hermelink et al. (Potato Research 31 (1988)241-246).

9. Determination of Glucose, Fructose and Sucrose

In order to determine the glucose, fructose and/or sucrose content,small pieces of potato tubers (with a diameter of approx. 10 mm) arefrozen in liquid nitrogen and subsequently extracted for 30 min at 80°C. in 0.5 ml 10 mM HEPES, pH 7.5; 80% (vol./vol.) ethanol. Thesupernatant containing the soluble components is withdrawn and thevolume is determined. The supernatant is used for determining the amountof soluble sugars. The quantitative determination of soluble glucose,fructose and sucrose is carried out in a reaction mixture with thefollowing composition:

100.0 mM imidazole/HCl, pH 6.9

1.5 mM MgCl₂

0.5 mM NADP⁺

1.3 mM ATP

10-50 μl sample

1.0 U glucose-6-phosphate dehydrogenase from yeast

The reaction mixture is incubated at room temperature for 5 minutes. Thesubsequent determination of sugars is carried out by means of standardphotometric methods by measuring the absorption at 340 nm aftersuccessive adding of

1.0 unit of hexokinase from yeast (for determining glucose)

1.0 unit of phosphoglucoisomerase from yeast (for determining fructose)and

1.0 unit of invertase from yeast (for determining sucrose).

EXAMPLE 1 The Isolation of Starch Granule-bound Proteins from PotatoStarch

The isolation of starch granule-bound proteins from potato starch hasbeen carried out by means of electroelution in an elution appliancewhich was constructed analogous to the “Model 422 Electro Eluter”(BIORAD Laboratories Inc., USA) but had a considerably greater volume(approx. 200 ml). 25 g dried starch were dissolved in elution buffer(final volume 80 ml). The starch was derived from potatoes which producean almost amylose-free starch due to the antisense-expression of a DNAsequence encoding the starch granule-bound starch synthase I (GBSS I)from potato. The suspension was heated to 70-80° C. in a water bath.Subsequently 72.07 g urea was added (final concentration 8 M) and thevolume was filled up to 180 ml with elution buffer. The starch dissolvedduring permanent stirring and acquired a paste-like consistency. Theproteins were electroeluted from the solution overnight by means of theelution appliance (100 V; 50-60 mA). The eluted proteins were carefullyremoved from the appliance. Suspended particles were removed in a briefcentrifugation. The supernatant was dialyzed at 4° C. 2 to 3 times forone hour against dialysis buffer. Subsequently, the volume of theprotein solution was determined. The proteins were precipitated byadding ammonium sulfate (final concentration 90%), which was done duringpermanent stirring at 0° C. The precipitated proteins were pelleted bycentrifugation and resuspended in protein buffer.

EXAMPLE 2 Identification and Isolation of cDNA Sequences Encoding StarchGranule-bound Proteins

The proteins isolated according to Example 1 were used for theproduction of polyclonal antibodies from rabbit, which specificallyrecognize starch granule-bound proteins.

By means of such antibodies a cDNA expression library was subsequentlyscreened for sequences encoding starch granule-bound proteins, usingstandard methods.

The Expression Library was Produced as Follows:

Poly (A⁺)-mRNA was isolated from potato tubers of the “Berolina”variety. Starting from the poly (A⁺)-mRNA, cDNA was produced accordingto the Gubler and Hoffmann method (Gene 25 (1983), 263-269), using anXho I-Oligo d(t)₁₈ primer. This cDNA was cut with Xho I after EcoRI-linker addition and ligated in an oriented manner in a lambda ZAP IIvector (Stratagene) cut with EcoR I and Xho I. Approximately 500,000plaques of a cDNA library constructed in such a way were screened forsequences which were recognized by polyclonal antibodies directedagainst starch granule-bound proteins.

In order to analyze the phage plaques these were transferred tonitrocellulose filters which had previously been incubated in a 10 mMIPTG solution for 30 to 60 minutes and had subsequently been dried onfilter paper. The transfer took place at 37° C. for 3 hours.Subsequently, the filters are incubated at room temperature for 30minutes in block reagent and washed for 5-10 minutes in TBST buffer. Thefilters were shaken with the polyclonal antibodies directed againststarch granule-bound proteins in a suitable dilution for one hour atroom temperature or for 16 hours at 4° C. The identification of plaquesexpressing a protein which was recognized by the polyclonal antibodieswas carried out by means of the “Blotting detection kit for rabbitantibodies RPN 23” (Amersham UK) according to the manufacturer'sinstructions.

Phage clones of the cDNA library expressing a protein which wasrecognized by the polyclonal antibodies were further purified by usingstandard methods.

By means of the in-vivo excision method, E. coli clones were obtainedfrom positive phage clones containing a double-stranded pBluescriptplasmid with the corresponding cDNA insertion. After checking the sizeand the restriction pattern of the insertions a suitable clone, pRL1,was further analyzed.

EXAMPLE 3 Sequence Analysis of the cDNA Insertion of the Plasmid pRL1

From an E. coli clone obtained according to Example 2 the plasmid pRL1was isolated and a part of the sequence of its cDNA insertion wasdetermined by standard procedures using the didesoxynucleotide method(Sanger et al., Proc. Natl. Acad. Sci. USA 74 (1977), 5463-5467). Theinsertion has a length of about 2450 bp. A part of the nucleotidesequence as well as the amino acid sequence derived therefrom isindicated under Seq ID No. 3 and under Seq ID No. 4.

A sequence analysis and a sequence comparison with known DNA sequencesshowed that the sequence indicated under Seq ID No. 3 is new andexhibits no significant homology to DNA sequences known so far.Moreover, the sequence analysis showed that the cDNA insertion is only apartial cDNA in which a part of the coding region at the 5′-end ismissing.

EXAMPLE 4 Identification and Isolation of a Complete cDNA Encoding aStarch Granule-bound Protein from Solanum Tuberosum

In order to isolate a complete cDNA corresponding to the partial cDNAinsertion of the plasmid pRL1, a further cDNA library was produced. Thiswas a guard-cell-specific cDNA library from Solanum tuberosum which wasconstructed as follows:

At first epidermis fragments from leaves of “Desirée” variety potatoplants were produced essentially according to the Hedrich et al. method(Plant Physiol. 89 (1989), 148), by harvesting approximately 60 leavesof six-weeks-old potato plants kept in the greenhouse. The center nervewas removed from the leaves. The leaves were subsequently crushed in abig “Waring blender” (with a volume of 1 liter) four times in cooled,distilled H₂O on the highest level for 15 seconds each. The suspensionwas filtered through a nylon sieve with a mesh size of 220 μm (Nybolt,Zurich, Switzerland) and washed in cold distilled water several times.The suspension itself was filtered through a 220 μm nylon sieve andintensely washed with cold distilled water. The residues (epidermisfragments) were crushed in a smaller “Waring blender” (with a volume of250 ml) four times in distilled water and ice on a lower level for 15seconds each. The suspension was filtered through a 220 μm nylon sieveand washed intensely with cold distilled water. The epidermis fragments(residues) were microscopically examined for contamination by mesophylcells. If contamination occurred the crushing step was repeated in asmall “Waring blender”.

The disruption of the guard cells of the epidermis fragments was carriedout by means of pulverizing in liquid nitrogen in a cooled mortar forapproximately two hours. In order to examine the disruption of the guardcells, probes were regularly taken and microscopically examined. Aftertwo hours, or if a sufficiently high amount of guard cells had beendisrupted, the obtained powder was filled into a reaction tube (with avolume of 50 ml) and resuspended in one volume GTC buffer (Chirgwin etal., Biochem. 18 (1979), 5294-5299). The suspension was centrifuged andthe supernatant was filtered through Miracloth (Calbiochem, La Jolla,Calif.). The filtrate was subjected to ultracentrifugation for 16 hours,as described in Glisin et al. (Biochemistry 13 (1974), 2633-2637) andMornex et al. (J. Clin. Inves. 77 (1986), 1952-1961). After thecentrifugation the RNA precipitate was dissolved in 250 μl GTC buffer.The RNA was precipitated by adding 0.05 volumes of 1 M acetic acid and0.7 volumes of ethanol. The RNA was precipitated by centrifugation andthe precipitate was washed with 3 M sodium acetate (pH 4.8) and 70%ethanol. The RNA was briefly dried and dissolved in DEPC treated water.

Poly A⁺-RNA was isolated from the isolated RNA according to standardmethods. Starting from the poly(A⁺)-mRNA, cDNA was produced according tothe Gubler and Hoffmann method (Gene 25 (1983), 263-269) by means of aXho I-oligo d(t)₁₈ primer. This cDNA was cut with Xho I after EcoRI-linker addition and ligated in an oriented manner in a lambda ZAP IIvector (Stratagene GmbH, Heidelberg, Germany) cut with EcoR I and Xho I.The packaging in phage heads was carried out using the Gigapack II Goldkit (Stratagene GmbH, Heidelberg, Germany) according to themanufacturer's instructions.

From such a cDNA library phage clones hybridizing with the cDNAinsertion of the pRL1 plasmid were isolated and purified according tostandard methods. By means of the in vivo excision method E. coli cloneswere obtained from positive phage clones containing a double-strandedpBluescript plasmid with the corresponding cDNA insertion. Afterchecking the size and the restriction pattern of the insertions,suitable clones were subjected to restriction mapping and sequenceanalysis. From a suitable clone the plasmid pRL2 (DSM 10225) wasisolated which contains a complete cDNA which encodes a starchgranule-bound protein from potato.

EXAMPLE 5 Sequence Analysis of the cDNA Insertion of the gRL2 Plasmid

The nucleotide sequence of the cDNA insertion of the pRL2 plasmid wasdetermined as described in Example 3. The insertion has a length of 4856bp. The nucleotide sequence as well as the amino acid sequence derivedtherefrom is indicated in Seq ID No. 1 and/or Seq ID No. 2. In thefollowing, the corresponding gene will be called RL-gene.

EXAMPLE 6 The Construction of the Plasmid P35S-anti-RL and theIntroduction of the Plasmid into the Genome of Potato Plants

By means of the restriction endonuclease Asp718 a DNA fragment with anapproximate length of 1800 bp was isolated from the pRL1 plasmid. Thiscorresponds to the DNA sequence indicated under Seq ID No. 3 andcontains a part of the open reading frame. The fragment was ligated intothe binary vector pBinAR cut with Asp718 (Höfgen and Willmitzer, PlantSci. 66 (1990), 221-230). This is a derivative of the binary vectorpBin19 (Bevan, Nucl. Acids Res. 12 (1984), 8711-8721). pBinAR wasconstructed as follows:

A fragment with a length of 529 bp comprising the nucleotides 6909-7437of the 35S promoter of the cauliflower-mosaic virus (Franck et al., Cell21 (1980), 285-294) was isolated from the plasmid pDH51 (Pietrzak etal., Nucl. Acids Res. 14, 5857-5868) as an EcoR I/Kpn I fragment andligated between the EcoR I and the Kpn I sites of the pBin19 polylinker.This led to the plasmid pBin19-A.

By means of the restriction-endonucleases Pvu II and Hind III a fragmentwith a length of 192 bp was isolated from the plasmid pAGV40(Herrera-Estrella et al., Nature 303, 209-213) comprising thepolyadenylation signal of gene 3 of the T-DNA of the Ti-plasmid pTiACH5(Gielen et al., EMBO J. 3, 835-846) (nucleotides 11749-11939). After theaddition of Sph I-linkers to the Pvu I site the fragment was ligatedbetween the Sph I and Hind III sites of pBin19-A. This led to plasmidpBinAR.

By means of restriction and sequence analysis recombinant vectors wereidentified in which the DNA fragment is inserted in the vector in such away that a part of the coding region of the cDNA insertion from pRL1 islinked with the 35S promoter in antisense orientation. The resultingplasmid p35S-anti-RL is shown in FIG. 1.

By inserting the cDNA fragment an expression cassette is produced whichconsists of the fragments A, B and C:

Fragment A (529 bp) contains the 35S promoter of the cauliflower-mosaicvirus (CaMV). The fragment comprises the nucleotides 6909 to 7437 of theCaMV (Franck et al., Cell 21 (1980), 285-294).

Apart from flanking regions, fragment B contains a part of theprotein-encoding areas of the cDNA insertion from plasmid pRL1. This wasisolated as an Asp718 fragment of pRL1 as described above and fused tothe 35S promoter in antisense orientation. Fragment C (192 bp) containsthe polyadenylation signal of gene 3 of the T-DNA of the Ti-plasmidpTiACH5 (Gielen et al., EMBO J. 3 (1984), 835-846).

The plasmid p35S-anti-RL has a size of approximately 12.8 kb. Theplasmid was transferred into potato plants by means ofAgrobacteria-mediated transformation, as described above. From thetransformed cells whole plants were regenerated. The transformed plantswere cultivated under greenhouse conditions. By analyzing total RNA in aNorthern Blot analysis concerning the disappearance of the transcriptscomplementary to the cDNA, the success of the genetic modification ofthe plants was assessed. For this purpose, total RNA was isolated fromleaves of transformed plants according to standard methods andsubsequently separated electrophoretically on an agarose gel. Then itwas transferred onto a nylon membrane and hybridized with aradioactively labelled probe having the sequence indicated under Seq IDNo. 1 or a part thereof. In about 5-10% of the transformed plants theband indicating the specific transcript under Seq ID No. 1 was missingin the Northern Blot analysis. The plants were used for analyzing thestarch quality.

EXAMPLE 7 The Construction of the Plasmid pB33-anti-RL and theIntroduction of the Plasmid into the Genome of Potato Plants

By means of the restriction endonuclease Asp718, a DNA fragment with anapproximate length of 1800 bp, which comprises a part of the openreading frame of the cDNA insertion was isolated from the plasmid pRL1and was ligated into the vector B33-Hyg which was cut with Asp718. Thisvector was constructed as follows:

The 35S promoter was removed from the pBinAR Hyg vector (DSM 9505) bymeans of the restriction endonucleases EcoR I and Asp718. A fragmentwith a length of about 1526 bp comprising the B33 promoter was isolatedfrom the plasmid p33-anti-BE (DSM 6146) by means of EcoR I and Asp718and inserted into the pBinAR Hyg vector (DSM 9505) cut with EcoR I andAsp718.

By inserting the cDNA fragment into the Asp718 site of the B33-Hygplasmid, an expression cassette is produced which consists of thefragments A, B and C as follows (FIG. 4):

Fragment A contains the B33 promoter from Solanum tuberosum (EP 3775092; Rocha-Sosa et al., EMBO J. 8 (1989), 23-29).

Apart from flanking regions, fragment B contains a part of the proteinencoding region of the cDNA insertion from the pRL1 plasmid. This wasisolated as an Asp718 fragment from pRL1 as described above and fused tothe B33 promoter in B33-Hyg in antisense orientation.

Fragment C (192 bp) contains the polyadenylation signal of gene 3 of theT-DNA of the Ti-plasmid pTiACH5 (Gielen et al., EMBO J. 3 (1984),835-846).

The plasmid pB33-anti-RL has a size of approximately 12.8 kb.

The plasmid was transferred into potato plants by means ofAgrobacteria-mediated transformation, as described above. From thetransformed cells whole plants were regenerated. The transformed plantswere cultivated under greenhouse conditions. By analyzing total RNA in aNorthern Blot analysis concerning the disappearance of the transcriptscomplementary to the cDNA the success of the genetic modification of theplants was assessed. For this purpose, total RNA was isolated fromtubers of transformed plants according to standard methods andsubsequently separated electrophoretically on an agarose gel. Then itwas transferred onto a nylon membrane and hybridized with aradioactively labelled probe showing the sequence indicated under Seq IDNo. 1 or a part thereof. In about 5-10% of the transformed plants theband indicating the transcript hybridizing with the cDNA of theinvention was missing in the Northern Blot Analysis. From these plantsstarch was isolated from tubers and analyzed as described in Example 8.

EXAMPLE 8 Analysis of the Transformed Potato Plants

The potato plants transformed according to Example 6 and Example 7 wereexamined with regard to the properties of the synthesized starch.Analyses were carried out with various lines of the potato plants whichhad been transformed with the plasmid p35S-anti-RL or the plasmidpB33-anti-RL and which in Northern Blot analysis had not exhibited theband indicating transcripts hybridizing to the DNA sequences of theinvention.

a) Determination of the Viscosity of Aqueous Solutions of the Starch

In order to determine the viscosity of the aqueous solutions of thestarch synthesized in transformed potato plants, starch was isolatedfrom tubers of plants which had been transformed with the plasmidp35S-anti-RL or the plasmid pB33-anti-RL using standard methods. 30 g ofstarch were each taken up in 450 ml H₂O and used for analysis in an Eviscograph (Brabender OHG Duisburg (Germany)). The appliance was usedaccording to the manufacturer's instructions. In order to determine theviscosity of the aqueous solution of the starch, the starch suspensionwas first heated from 50° C. to 96° C. at a speed of 3° C. per minute.The temperature was subsequently kept at 96° C. for min. The solutionwas then cooled from 96° C. to 50° C. at a speed of 3° C. per minute.During the whole process the viscosity was determined. Representativeresults of such measurements are set forth in the form of graphs inFIGS. 3, 4 and 5, in which the viscosity is. shown depending on time.FIG. 3 shows a typical Brabender graph for starch isolated fromwildtype-plants of the potatoe variety Désirée. FIGS. 4 and 5 show atypical Brabender graph for starch isolated from potato plants which hadbeen transformed with the plasmid p35S-anti-RL or pB33-anti-RL. Fromthese graphs characteristic values may be deduced.

The characteristic values for wildtype-plants are as follows:

TABLE 1 Time Torque Temperature Value [min:sec] [BE] [° C.] A  6:30 60.5 ± 17.7 69.9 ± 0.57 B 11:30  1838.0 ± 161.2 86.0 ± 2.1  C 15:151412.0 ± 18.4 96.0 D 45:15  526.0 ± 17.0 96.0 E 60:30 812.0 ± 8.5 50.0 F70:45 853.0 ± 5.7 50.0

The values represent the average values obtained from two differentmeasurements.

In Table 1 and the following Tables 2 and 3 the abbreviations signifythe following:

A: start of pastification

B: maximum viscosity

C: start of 96° C. period

D: start of cooling-off time

E: end of cooling-off time

F: end of the end-50° C. period

For plants which had been transformed with the plasmid p35S-anti-RL(line P2), the characteristic values are the following:

TABLE 2 Time Torque Temperature Value [min:sec] [BE] [° C.] A  6:00 50.069.0 B 14:00 820.0 93.0 C 15:15 815.0 96.0 D 45:15 680.0 96.0 E 60:301150.0 50.0 F 70:45 1200.0 50.0

For plants which had been transformed with the plasmid pB33-anti-RL(line P3), the characteristic values are the following:

TABLE 3 Time Torque Temperature Value [min:sec] [BE] [° C.] A 7:0 31.071.0 B 12:45 671.0 88.3 C 15:15 662.0 96.0 D 45:15 607.0 96.0 E 60:301063.0 50.0 F 70:45 1021.0 50.0

FIGS. 3, 4 and 5 explicitly show that the starch obtained fromtransformed plants differs from starch from wildtype plants particularlyin that the viscosity increases only very slightly during heating. Thus,during heating the maximum viscosity of the modified starch fromtransformed plants is more than 50% lower than in the case of wildtypestarch.

During cooling, on the other hand, the viscosity of the starch isolatedfrom transformed plants increases more than in the case ofwildtype-plants.

b) Determination of the Phosphate Content of the Starch

The phosphate content of the starch was determined by measuring theamount of phosphate bound to the C-6-position of the glucose residues.For this purpose, starch was first degraded by acid hydrolysis and theglucose-6-phosphate content was subsequently determined by means of anenzyme test, as described in the following.

100 mg starch were incubated in 500 μl 0.7 N HCl for 4 hours at 100° C.After acid hydrolysis 10 μl of the reaction were added to 600 μlimidazole buffer (100 mM imidazole, 5 mM MgCl₂, pH 6.9, 0.4 mM NAD⁺).The amount of glucose-6-phosphate in the reaction mixture was determinedby conversion with the enzyme glucose-6-phosphate-dehydrogenase. Forthis purpose, 1 U glucose-6-phosphate-dehydrogenase (from Leuconostocmesenteroides (Boehringer Mannheim)) was added to the reaction mixtureand the amount of produced NADH was determined by measuring theabsorption at 340 nm.

The glucose-6-phosphate content of 1 mg starch is indicated in thefollowing table for non-transformed potato plants of the variety Désiréeas well as for two lines (P1 (35S-anti-RL); P2(35S-anti-RL)) oftransgenic potato plants which had been transformed with the plasmidp35S-anti-RL.

TABLE 4 Plants nmol glucose-6-phosphate/mg starch % Wildtype 12.89 ±1.34  100 P1 (35S-anti-RL) 2.25 ± 0.41 17.4 P2 (35S-anti-RL) 1.25 ± 0  9.7

The following table shows the glucose-6-phosphate content per milligramstarch in potato plants which were transformed with the plasmidpB33-anti-RL, compared to starch from non-transformed plants (S.tuberosum c.v. Désirée).

TABLE 5 Plants nmol glucose-6-phosphate/mg starch % Wildtype 9.80 ± 0.68100  7 4.50 ± 0.73 45.9 37 2.64 ± 0.99 26.9 45 1.14 ± 0.44 11.6 31 1.25± 0.49 12.8

The plants 7, 37, 45 and 31 represent independent transformants whichhad been transformed with the plasmid pB33-anti-RL. Plant 37 representsline P3 for which a Brabender graph is plotted in FIG. 5.

The values show that the phosphate content of the modified starch fromtransgenic potato plants is at least 50% lower when compared to starchfrom wildtype plants.

c) Determination of Glucose, Fructose and Sucrose Content of TubersAfter Storage at 4° C.

Tubers of plants from various transgenic lines which had beentransformed with the antisense-construct p35S-anti-RL as well as tubersof wildtype plants were stored at 4° C. or, respectively, at 20° C. indarkness, for two months. Subsequently, the amounts of glucose, fructoseand sucrose were determined as described above. For two transgenic linesthe representative values obtained were the following:

TABLE 6 Glucose Fructose Sucrose 20° C. 4° C. 20° C. 4° C. 20° C. 4° C.Wildtype 0.84 55.4 0.62 52.8 8.5 13.1 cv Désirée Transgenic 1.12 6.70.75 7.8 7.5 10.1 line 15 Transgenic 1.00 6.4 0.75 7.5 6.9 6.9 line 11The values in the table are indicated in μmol hexose or sucrose/g freshweight.

From the values of Table 6 it becomes obvious that the accumulation ofreducing sugars in the tubers is considerably lower in transgenic plantsstored at 4° C. than in wildtype plants.

Altogether the modified starch isolated from transgenic potato plantsresembles starch from maize-wildtype plants. However, in comparison ithas the advantage that its taste is neutral and that it is thereforemore suitable for various uses in the foodstuffs area.

EXAMPLE 9 Expression of the cDNA Insertion of the pRL2 Plasmid in E.coli

(a) Transformation of Bacterial Cells

In order to express the cDNA insertion of the plasmid pRL2 the cells ofthe E. coli strain DH5α are first transformed with the pACAC plasmid.This plasmid contains a DNA fragment encoding theADP-glucose-pyrophosphorylase (AGPase) from E. coli, under the controlof the lac Z promoter. The fragment had been isolated from the vectorpEcA-15 as a DraI/HaeII fragment with a size of about 1.7 kb (see B.Müller-Röber (1992), dissertation, FU Berlin) and after filling in itssticky ends it had been cloned into a pACAC184 vector linearized withHindIII. The expression of AGPase is to cause an increase of theglycogen synthesis in transformed E. coli cells. The cells transformedin such a way will in the following be named E. coli-K1-cells.

In order to determine the enzyme activity of the protein encoded by thecDNA of plasmid pRL2, E. coli-K1-cells were transformed with the pRL2plasmid. The transformed E. coli cells which contain the pACAC plasmidas well as the pRL2 plasmid will in the following be named E.coli-K2-cells.

The transfer of the plasmid DNA into the bacterial cells was carried outaccording to the Hanahan method (J. Mol. Biol. 166 (1983), 557-580). Thetransformed E. coli cells were plated onto agar culture dishes with thefollowing composition:

YT medium containing 1, 5% Bacto agar 50 mM sodium phosphate buffer, pH7.2 1% glucose 10 μg/ml chloramphenicol in the case of E. coli-K1-cellsor 10 μg/ml chloramphenicol and 10 μg/ml ampicillin in the case of E.coli-K2-cells.

Escherichia coli cells of the DH5α strain which had been transformedwith the plasmid pRL2+pACAC (E. coli-K2-cells) and also—forcontrol—solely with the pACAC plasmid (E. coli-K1-cells), were raised onagar plates. The formed glycogen of the various cultures was examinedwith respect to the degree of phosphorylization (at the C-6 position ofthe glucose molecule), as described in the following.

(b) Isolation of Bacterial Alycogen

In order to isolate bacterial glycogen, the bacteria colony which hadgrown after transformation was floated from each 6 agar plates (Ø 135mm) with 5 ml YT medium for each plate. The bacterial suspension wascentrifuged at 4500×g for 5 minutes. The bacterial precipitate wasresuspended in 10 ml YT medium. Disruption of the bacteria was carriedout by adding 2 volumes of disruption medium (0.2 N NaOH; 1% SDS) and byincubation at room temperature for 5 minutes. By adding 3 volumes ofEtOH abs., incubating at 4° C. for minutes and subsequent centrifugingat 8000×g for 15 minutes, the glycogen was sedimented. Then theprecipitate was washed with 100 ml of 70% EtOH and again sedimented bymeans of a centrifugation step (10 minutes at 8000×g). The washingprocedure was repeated four times.

(c) Determination of the Total Glycogen Content

The isolated and sedimented glycogen was first degraded into singleglucose molecules by means of acidic hydrolysis (dissolving of theprecipitate in 2 ml 0.7 N HCl; incubation for 4 hours at 100° C.). Theglucose content of the solution was determined by means of coupledenzymatic reaction of a starch test with a photometer (Kontron) at awave length of 340 nm according to the manufacturer's (BoehringerMannheim) instructions.

The reaction buffer contains: 100 mM MOPS, pH 7.5 10 mM MgCl₂ 2 mM EDTA0.25 mM NADP 1 mM ATP 1 U/ml glucose-6-phosphate- dehydrogenase 2 U/mlhexokinase

Die measurement was carried out at 25° C. with 10 μl glucose solution.

(d) Determination of the Glucose-6-Phosphate Content

In order to determine the content of glucose molecules phosphorylated atthe C-6 position, equal amounts of glucose of the various bacterialcultures were used. By adding the same volumes of 0.7 N KOH to theglycogens degraded into its glucose molecules by acidic hydrolysis (asabove), the solution was neutralized.

The reaction buffer contains: 100 mM MOPS, pH 7.5 10 mM MgCl₂ 2 mM EDTA0.25 mM NADP 2 U/ml glucose-6-phosphate- dehydrogenase

The measurement was carried out at 25° C. with 100 to 150 μl glucosesolution.

(e) Identification of an Enzyme Activity Phosphorylating BacterialGlycogen

The results of the determination of the phosphate content of theglycogen synthesized in the bacterial cells show that the glycogen ofthe E. coli cells, which had been transformed with the pACAC+pRL2plasmids, exhibits a 290±25% increased phosphorylation at the C-6position of the glucose when comparing with the control reaction (E.coli cells transformed with the PACYC plasmid) (see the followingtable).

E. coli cells glucose-6-phosphase: glucose in glycogen E. coli-K11:(4600 ± 1150) E. coli-K2 1:(1570 ± 390)

The degrees of phosphorylation indicated herein are the average value ofat least 6 measurements starting from 6 independent transformations andglycogen isolations.

EXAMPLE 10 Integration of the Plasmid p35S-anti-RL in Combination withthe Plasmid p35SH-anti-BE into the Genome of Potato Plants

The plasmid p35S-anti-RL was constructed as described in Example 6. Theplasmid p35SH-anti-BE was constructed as described in the applicationWO95/07355, Example 3. Both plasmids were sequentially transferred intopotato plants by means of the Agrobacterium mediated transformation asdescribed above. For this purpose, the plasmid p35SH-anti-BE was firsttransformed in potato plants. Whole plants were regenerated and selectedfor a reduced expression of the branching enzyme gene. Subsequently, theplasmid p35S-anti-RL was transformed into the transgenic plants alreadyshowing a reduced expression of the branching enzyme. From thetransformed cells transgenic plants were again regenerated and thetransformed plants were cultivated under greenhouse conditions. Byanalyzing total RNA in an RNA Blot analysis with respect to thedisappearance of the transcripts complementary to the branching enzymecDNA or the RL cDNA, the success of the genetic modification of theplants with respect to a highly reduced expression of the branchingenzyme gene as well as with respect to a highly reduced expression ofthe RL gene was assessed. For this purpose, total RNA was isolated fromleaves of transformed plants according to the described methods andsubsequently separated by means of gel electrophoresis, transferred ontoa membrane, hybridized with a radioactively labelled probe showing thesequence indicated under Seq ID No. 1 or a part thereof and thenhybridized with a radioactively labelled probe showing the sequence ofthe branching enzyme cDNA (cf. WO92/14827, Example 1) or a part thereof.In about 5%-10% of the transformed plants the band indicating thespecific transcript of the sequence indicated under Seq ID No. 1 as wellas the band indicating the specific transcript of the branching enzymecDNA (cf. WO92/14827) was missing in the RNA Blot analysis. Theseplants, which were designated R4 plants were used for analyzing thequality of the starch contained in tubers.

EXAMPLE 11 Integration of the Plasmid pB33-anti-RL in Combination withthe Plasmid pB33-anti-GBSSI into the Genome of Potato Plants

The plasmid pB33-anti-RL was constructed as described in Example 7. Theplasmid pB33-anti-GBSSI was constructed as follows:

The DraI/DraI fragment of the promoter region of the patatin class Igene B33 from Solanum tuberosum comprising the nucleotides −1512 to +14(Rocha-Sosa et al., EMBO J 8 (1989), 23-29) was ligated into the SmaIsite of the pUC19 plasmid. From the resulting plasmid the promoterfragment was ligated into the polylinker region of the pBin19 plasmid(Bevan, Nucleic Acids Research 12 (1984), 8711-8721) as an EcoRI/HindIIIfragment. Subsequently, the 3′ EcoRI fragment 1181 to 2511 of the GBSSIgene of Solanum tuberosum (Hergersberg, dissertation (1988), Universityof Cologne) was ligated into the EcoRI site of the resulting plasmid.

Both plasmids were transferred sequentially into potato plants by meansof Agrobacterium mediated transformation as described in Example 10.From the transformed cells whole plants were regenerated and thetransformed plants were cultivated under greenhouse conditions. Byanalyzing the complete RNA in a RNA Blot analysis with regard to thedisappearance of the transcripts complementary to the two cDNAs, thesuccess of the genetic modification of the plants was assessed. For thispurpose, total RNA was isolated from tubers of transformed plantsaccording to standard methods and subsequently separated on agarose gelby means of gel electrophoresis, transferred onto a membrane andhybridized with a radioactively labelled probe showing the sequenceindicated under Seq ID No. 1 or a part thereof. Afterwards, the samemembrane was hybridized with a radioactively labelled probe having thesequence of the GBSSI gene or a part of this sequence (Hergersberg,dissertation (1988) University of Cologne). In about 5%-10% of thetransformed plants the band indicating the transcripts hybridizing tothe cDNA of the invention or the GBSSI cDNA were missing in the RNA Blotanalysis. From the tubers of these plants, which were designated R3plants, starch was isolated and analyzed.

EXAMPLE 12 Starch Analysis of R4 Plants

The potato plants transformed according to Example 10 were examined withrespect to the properties of the synthesized starch. The analyses werecarried out with various lines of the potato plants which had beentransformed with the plasmids p35S-anti-RL and p35SH-anti-BE and whichdid no longer—or only in extremely reduced form—show the bandsindicating transcripts hybridizing to the DNA sequences of the inventionor to the sequence of the branching cDNA in RNA Blot analysis.

a) Determination of the Viscosity of Aqueous Solutions of the Starch

In order to determine the viscosity of the aqueous solutions of thestarch synthesized in transformed potato plants, starch was isolatedfrom tubers of plants which had been transformed with the plasmidp35S-anti-RL and the plasmid p35SH-anti-BE using standard methods. 2 gof starch were each dissolved in 25 ml H₂O and used for analysis with aRapid Visco Analyser (Newport Scientific Pty Ltd, Investment SupportGroup, Warriewood NSW 2102, Australia). The equipment was used accordingto the instructions of the manufacturer. In order to determine theviscosity of the aqueous solution of the starch, the starch suspensionwas first heated from 50° C. to 95° C. with a speed of 12° C. perminute. The temperature was then kept at 95° C. for 2.5 minutes.Afterwards, the solution was cooled from 95° C. to 50° C. with a speedof 12° C. per minute. During the whole process the viscosity wasmeasured. Representative results of such measurements are set forth inthe form of graphs in which the viscosity is shown depending on time.FIG. 6 shows a typical RVA graph for starch isolated from thewildtype-plants of potato of the variety Désirée. Lines 2 and 3 show atypical RVA graph for starch isolated from the tubers of plants whichhad been transformed with the plasmid p35SH-anti-BE and with the plasmidp35S-anti-RL, respectively. Line 4 shows a typical RVA graph for starchisolated from tubers of plants which had been transformed with plasmidp35SH-anti-BE in combination with plasmid p35S-anti-RL. Line 4 ischaracterized in that there is no temperature-dependent increase ofviscosity.

b) Determination of the Amylose/Amylopectin Ratio

Starch which was isolated from the tubers of transformed potato plantswas examined with respect to the ratio of amylose to amylopectin. Theplant line R4-1 (shown in line 4 of FIG. 6) exhibited an amylose contentof more than 70%. For the plant line R4-3 an amylose value of 27% wasmeasured, whereas the amylose content in wildtype starch of the Désiréevariety ranges between 19 and 22%.

EXAMPLE 13 Starch Analysis of R3 Plants

The potato plants transformed according to Example 11 were examined withrespect to the properties of the synthesized starch. The analyses werecarried out with various lines of the potato plants which had beentransformed with the plasmids pB33-anti-RL and pB33-anti-GBSSI and whichdid no longer—or only in extremely reduced form—show the bandsindicating transcripts hybridizing to the DNA sequences of the inventionor to the sequence of the GBSSI cDNA in RNA Blot analysis.

a) Determination of the Viscosity of Aqueous Solutions of the Starch

In order to determine the viscosity of the aqueous solution of thestarch synthesized in transformed potato plants, starch was isolatedfrom tubers of plants which had been transformed with the plasmidpB33-anti-RL in combination with the plasmid pB33-anti-GBSSI usingstandard methods. The viscosity was determined by means of a Rapid ViscoAnalyser according to the method described in Example 12, part a. Theresults are indicated in FIG. 7. In line 1, FIG. 7 shows a typical RVAgraph for starch isolated from the wildtype-plants of the Désirée potatovariety. Lines 2 and 3 show typical RVA graphs for starches isolatedfrom potato plants which had been transformed with the plasmidpB33-anti-GBSSI and with the plasmid p35S-anti-RL, respectively. Line 4shows a typical RVA graph for starch isolated from potato plants whichhad been transformed with the plasmid pB33-anti-GBSSI in combinationwith the plasmid pB33-anti-RL. This graph is characterized in that themaximum viscosity and the increase of viscosity at 50° C. are missing.Furthermore, this starch is characterized in that the glue obtainedafter RVA treatment exhibits almost no retrogradation after incubatingat room temperature for several days.

b) Determination of the Amylose/Amylopectin Ratio

Starch which was isolated from the tubers of transformed potato plantswas examined with respect to the ratio of amylose to amylopectin. Theplant line R3-5 (shown in line 4 of FIG. 7) exhibited an amylose contentof less than 4%. For the plant line R3-6 an amylose content of less than3% was measured. The amylose content in wildtype starch of the Désiréevariety ranges between 19 and 22%.

c) Determination of the Phosphate Content of Starch

The phosphate content of the starch was determined by measuring theamount of phosphate bound to the C-6-position of the glucose residues.For this purpose, starch was first degraded by acid hydrolysis and theglucose-6-phosphate content was subsequently determined by means of anenzyme test, as described in the following.

100 mg starch were incubated in 500 μl 0.7 N HCl for 4 hours at 100° C.After acid hydrolysis 10 μl of the reaction mixture were added to 600 μlimidazole buffer (100 mM imidazole, 5 mM MgCl₂, pH 6.9, 0.4 mM NAD⁺).The amount of glucose-6-phosphate in the preparation is determined byconversion with the enzyme glucose-6-phosphate-dehydrogenase. For thispurpose, 1 U glucose-6-phosphate-dehydrogenase (from Leuconostocmesenteroides (Boehringer Mannheim)) was added to the reaction mixtureand the amount of produced NADH was determined by measuring theabsorption at 340 nm.

The glucose-6-phosphate content per 1 mg starch is indicated in thefollowing table for non-transformed potato plants of the variety Désiréeas well as for the R3-5 and the R3-6 line of transgenic potato plantswhich had been transformed with the plasmid pB33-anti-RL in combinationwith the plasmid pB33-anti-GBSSI. As a comparison, the value of thestarch from the so-called waxy potato (US2-10) which had beentransformed with the plasmid pB33-anti-GBSSI, is also indicated.

TABLE 7 Plants nmol glucose-6-phosphate/mg starch % Wildtype 9.80 ± 0.68100 R3-5 1.32 ± 0.10 13 R3-6 1.37 ± 0.15 14 US2-10 10.82 ± 0.42  110

4 4856 base pairs nucleotide single linear cDNA to mRNA Solanumtuberosum C.V. Berolina CDS 105..4497 1 CATCTTCATC GAATTTCTCG AAGCTTCTTCGCTAATTTCC TGGTTTCTTC ACTCAAAATC 60 GACGTTTCTA GCTGAACTTG AGTGAATTAAGCCAGTGGGA GGAT ATG AGT AAT TCC 116 Met Ser Asn Ser 1 TTA GGG AAT AACTTG CTG TAC CAG GGA TTC CTA ACC TCA ACA GTG TTG 164 Leu Gly Asn Asn LeuLeu Tyr Gln Gly Phe Leu Thr Ser Thr Val Leu 5 10 15 20 GAA CAT AAA AGTAGA ATC AGT CCT CCT TGT GTT GGA GGC AAT TCT TTG 212 Glu His Lys Ser ArgIle Ser Pro Pro Cys Val Gly Gly Asn Ser Leu 25 30 35 TTT CAA CAA CAA GTGATC TCG AAA TCA CCT TTA TCA ACT GAG TTT CGA 260 Phe Gln Gln Gln Val IleSer Lys Ser Pro Leu Ser Thr Glu Phe Arg 40 45 50 GGT AAC AGG TTA AAG GTGCAG AAA AAG AAA ATA CCT ATG GAA AAG AAG 308 Gly Asn Arg Leu Lys Val GlnLys Lys Lys Ile Pro Met Glu Lys Lys 55 60 65 CGT GCT TTT TCT AGT TCT CCTCAT GCT GTA CTT ACC ACT GAT ACC TCT 356 Arg Ala Phe Ser Ser Ser Pro HisAla Val Leu Thr Thr Asp Thr Ser 70 75 80 TCT GAG CTA GCA GAA AAG TTC AGTCTA GGG GGG AAT ATT GAG CTA CAG 404 Ser Glu Leu Ala Glu Lys Phe Ser LeuGly Gly Asn Ile Glu Leu Gln 85 90 95 100 GTT GAT GTT AGG CCT CCC ACT TCAGGT GAT GTG TCC TTT GTG GAT TTT 452 Val Asp Val Arg Pro Pro Thr Ser GlyAsp Val Ser Phe Val Asp Phe 105 110 115 CAA GTA ACA AAT GGT AGT GAT AAACTG TTT TTG CAC TGG GGG GCA GTA 500 Gln Val Thr Asn Gly Ser Asp Lys LeuPhe Leu His Trp Gly Ala Val 120 125 130 AAA TTC GGG AAA GAA ACA TGG TCTCTT CCG AAT GAT CGT CCA GAT GGG 548 Lys Phe Gly Lys Glu Thr Trp Ser LeuPro Asn Asp Arg Pro Asp Gly 135 140 145 ACC AAA GTG TAC AAG AAC AAA GCACTT AGA ACT CCA TTT GTT AAA TCT 596 Thr Lys Val Tyr Lys Asn Lys Ala LeuArg Thr Pro Phe Val Lys Ser 150 155 160 GGC TCT AAC TCC ATC CTG AGA CTGGAG ATA CGA GAC ACT GCT ATC GAA 644 Gly Ser Asn Ser Ile Leu Arg Leu GluIle Arg Asp Thr Ala Ile Glu 165 170 175 180 GCT ATT GAG TTT CTC ATA TACGAT GAA GCC CAC GAT AAA TGG ATA AAG 692 Ala Ile Glu Phe Leu Ile Tyr AspGlu Ala His Asp Lys Trp Ile Lys 185 190 195 AAT AAT GGT GGT AAT TTT CGTGTC AAA TTG TCA AGA AAA GAG ATA CGA 740 Asn Asn Gly Gly Asn Phe Arg ValLys Leu Ser Arg Lys Glu Ile Arg 200 205 210 GGC CCA GAT GTT TCT GTT CCTGAG GAG CTT GTA CAG ATC CAA TCA TAT 788 Gly Pro Asp Val Ser Val Pro GluGlu Leu Val Gln Ile Gln Ser Tyr 215 220 225 TTG AGG TGG GAG AGG AAG GGAAAA CAG AAT TAC CCC CCT GAG AAA GAG 836 Leu Arg Trp Glu Arg Lys Gly LysGln Asn Tyr Pro Pro Glu Lys Glu 230 235 240 AAG GAG GAA TAT GAG GCT GCTCGA ACT GTG CTA CAG GAG GAA ATA GCT 884 Lys Glu Glu Tyr Glu Ala Ala ArgThr Val Leu Gln Glu Glu Ile Ala 245 250 255 260 CGT GGT GCT TCC ATA CAGGAC ATT CGA GCA AGG CTA ACA AAA ACT AAT 932 Arg Gly Ala Ser Ile Gln AspIle Arg Ala Arg Leu Thr Lys Thr Asn 265 270 275 GAT AAA AGT CAA AGC AAAGAA GAG CCT CTT CAT GTA ACA AAG AGT GAT 980 Asp Lys Ser Gln Ser Lys GluGlu Pro Leu His Val Thr Lys Ser Asp 280 285 290 ATA CCT GAT GAC CTT GCCCAA GCA CAA GCT TAC ATT AGG TGG GAG AAA 1028 Ile Pro Asp Asp Leu Ala GlnAla Gln Ala Tyr Ile Arg Trp Glu Lys 295 300 305 GCA GGA AAG CCG AAC TATCCT CCA GAA AAG CAA ATT GAA GAA CTC GAA 1076 Ala Gly Lys Pro Asn Tyr ProPro Glu Lys Gln Ile Glu Glu Leu Glu 310 315 320 GAA GCA AGA AGA GAA TTGCAA CTT GAG CTT GAG AAA GGC ATT ACC CTT 1124 Glu Ala Arg Arg Glu Leu GlnLeu Glu Leu Glu Lys Gly Ile Thr Leu 325 330 335 340 GAT GAG TTG CGG AAAACG ATT ACA AAA GGG GAG ATA AAA ACT AAG GTG 1172 Asp Glu Leu Arg Lys ThrIle Thr Lys Gly Glu Ile Lys Thr Lys Val 345 350 355 GAA AAG CAC CTG AAAAGA AGT TCT TTT GCC GTT GAA AGA ATC CAA AGA 1220 Glu Lys His Leu Lys ArgSer Ser Phe Ala Val Glu Arg Ile Gln Arg 360 365 370 AAG AAG AGA GAC TTTGGG CAT CTT ATT AAT AAG TAT ACT TCC AGT CCT 1268 Lys Lys Arg Asp Phe GlyHis Leu Ile Asn Lys Tyr Thr Ser Ser Pro 375 380 385 GCA GTA CAA GTA CAAAAG GTC TTG GAA GAA CCA CCA GCC TTA TCT AAA 1316 Ala Val Gln Val Gln LysVal Leu Glu Glu Pro Pro Ala Leu Ser Lys 390 395 400 ATT AAG CTG TAT GCCAAG GAG AAG GAG GAG CAG ATT GAT GAT CCG ATC 1364 Ile Lys Leu Tyr Ala LysGlu Lys Glu Glu Gln Ile Asp Asp Pro Ile 405 410 415 420 CTA AAT AAA AAGATC TTT AAG GTC GAT GAT GGG GAG CTA CTG GTA CTG 1412 Leu Asn Lys Lys IlePhe Lys Val Asp Asp Gly Glu Leu Leu Val Leu 425 430 435 GTA GCA AAG TCCTCT GGG AAG ACA AAA GTA CAT CTA GCT ACA GAT CTG 1460 Val Ala Lys Ser SerGly Lys Thr Lys Val His Leu Ala Thr Asp Leu 440 445 450 AAT CAG CCA ATTACT CTT CAC TGG GCA TTA TCC AAA AGT CCT GGA GAG 1508 Asn Gln Pro Ile ThrLeu His Trp Ala Leu Ser Lys Ser Pro Gly Glu 455 460 465 TGG ATG GTA CCACCT TCA AGC ATA TTG CCT CCT GGG TCA ATT ATT TTA 1556 Trp Met Val Pro ProSer Ser Ile Leu Pro Pro Gly Ser Ile Ile Leu 470 475 480 GAC AAG GCT GCCGAA ACA CCT TTT TCA GCC AGT TCT TCT GAT GGT CTA 1604 Asp Lys Ala Ala GluThr Pro Phe Ser Ala Ser Ser Ser Asp Gly Leu 485 490 495 500 ACT TCT AAGGTA CAA TCT TTG GAT ATA GTA ATT GAA GAT GGC AAT TTT 1652 Thr Ser Lys ValGln Ser Leu Asp Ile Val Ile Glu Asp Gly Asn Phe 505 510 515 GTG GGG ATGCCA TTT GTT CTT TTG TCT GGT GAA AAA TGG ATT AAG AAC 1700 Val Gly Met ProPhe Val Leu Leu Ser Gly Glu Lys Trp Ile Lys Asn 520 525 530 CAA GGG TCGGAT TTC TAT GTT GGC TTC AGT GCT GCA TCC AAA TTA GCA 1748 Gln Gly Ser AspPhe Tyr Val Gly Phe Ser Ala Ala Ser Lys Leu Ala 535 540 545 CTC AAG GCTGCT GGG GAT GGC AGT GGA ACT GCA AAG TCT TTA CTG GAT 1796 Leu Lys Ala AlaGly Asp Gly Ser Gly Thr Ala Lys Ser Leu Leu Asp 550 555 560 AAA ATA GCAGAT ATG GAA AGT GAG GCT CAG AAG TCA TTT ATG CAC CGG 1844 Lys Ile Ala AspMet Glu Ser Glu Ala Gln Lys Ser Phe Met His Arg 565 570 575 580 TTT AATATT GCA GCT GAC TTG ATA GAA GAT GCC ACT AGT GCT GGT GAA 1892 Phe Asn IleAla Ala Asp Leu Ile Glu Asp Ala Thr Ser Ala Gly Glu 585 590 595 CTT GGTTTT GCT GGA ATT CTT GTA TGG ATG AGG TTC ATG GCT ACA AGG 1940 Leu Gly PheAla Gly Ile Leu Val Trp Met Arg Phe Met Ala Thr Arg 600 605 610 CAA CTGATA TGG AAC AAA AAC TAT AAC GTA AAA CCA CGT GAA ATA AGC 1988 Gln Leu IleTrp Asn Lys Asn Tyr Asn Val Lys Pro Arg Glu Ile Ser 615 620 625 AAG GCTCAG GAC AGA CTT ACA GAC TTG TTG CAG AAT GCT TTC ACC AGT 2036 Lys Ala GlnAsp Arg Leu Thr Asp Leu Leu Gln Asn Ala Phe Thr Ser 630 635 640 CAC CCTCAG TAC CGT GAA ATT TTG CGG ATG ATT ATG TCA ACT GTT GGA 2084 His Pro GlnTyr Arg Glu Ile Leu Arg Met Ile Met Ser Thr Val Gly 645 650 655 660 CGTGGA GGT GAA GGG GAT GTA GGA CAG CGA ATT AGG GAT GAA ATT TTG 2132 Arg GlyGly Glu Gly Asp Val Gly Gln Arg Ile Arg Asp Glu Ile Leu 665 670 675 GTCATC CAG AGG AAC AAT GAC TGC AAG GGT GGT ATG ATG CAA GAA TGG 2180 Val IleGln Arg Asn Asn Asp Cys Lys Gly Gly Met Met Gln Glu Trp 680 685 690 CATCAG AAA TTG CAT AAT AAT ACT AGT CCT GAT GAT GTT GTG ATC TGT 2228 His GlnLys Leu His Asn Asn Thr Ser Pro Asp Asp Val Val Ile Cys 695 700 705 CAGGCA TTA ATT GAC TAC ATC AAG AGT GAT TTT GAT CTT GGT GTT TAT 2276 Gln AlaLeu Ile Asp Tyr Ile Lys Ser Asp Phe Asp Leu Gly Val Tyr 710 715 720 TGGAAA ACC CTG AAT GAG AAC GGA ATA ACA AAA GAG CGT CTT TTG AGT 2324 Trp LysThr Leu Asn Glu Asn Gly Ile Thr Lys Glu Arg Leu Leu Ser 725 730 735 740TAT GAC CGT GCT ATC CAT TCT GAA CCA AAT TTT AGA GGA GAT CAA AAG 2372 TyrAsp Arg Ala Ile His Ser Glu Pro Asn Phe Arg Gly Asp Gln Lys 745 750 755GGT GGT CTT TTG CGT GAT TTA GGT CAC TAT ATG AGA ACA TTG AAG GCA 2420 GlyGly Leu Leu Arg Asp Leu Gly His Tyr Met Arg Thr Leu Lys Ala 760 765 770GTT CAT TCA GGT GCA GAT CTT GAG TCT GCT ATT GCA AAC TGC ATG GGC 2468 ValHis Ser Gly Ala Asp Leu Glu Ser Ala Ile Ala Asn Cys Met Gly 775 780 785TAC AAA ACT GAG GGA GAA GGC TTT ATG GTT GGA GTC CAG ATA AAT CCT 2516 TyrLys Thr Glu Gly Glu Gly Phe Met Val Gly Val Gln Ile Asn Pro 790 795 800GTA TCA GGC TTG CCA TCT GGC TTT CAG GAC CTC CTC CAT TTT GTC TTA 2564 ValSer Gly Leu Pro Ser Gly Phe Gln Asp Leu Leu His Phe Val Leu 805 810 815820 GAC CAT GTG GAA GAT AAA AAT GTG GAA ACT CTT CTT GAG AGA TTG CTA 2612Asp His Val Glu Asp Lys Asn Val Glu Thr Leu Leu Glu Arg Leu Leu 825 830835 GAG GCT CGT GAG GAG CTT AGG CCC TTG CTT CTC AAA CCA AAC AAC CGT 2660Glu Ala Arg Glu Glu Leu Arg Pro Leu Leu Leu Lys Pro Asn Asn Arg 840 845850 CTA AAG GAT CTG CTG TTT TTG GAC ATA GCA CTT GAT TCT ACA GTT AGA 2708Leu Lys Asp Leu Leu Phe Leu Asp Ile Ala Leu Asp Ser Thr Val Arg 855 860865 ACA GCA GTA GAA AGG GGA TAT GAA GAA TTG AAC AAC GCT AAT CCT GAG 2756Thr Ala Val Glu Arg Gly Tyr Glu Glu Leu Asn Asn Ala Asn Pro Glu 870 875880 AAA ATC ATG TAC TTC ATC TCC CTC GTT CTT GAA AAT CTC GCA CTC TCT 2804Lys Ile Met Tyr Phe Ile Ser Leu Val Leu Glu Asn Leu Ala Leu Ser 885 890895 900 GTG GAC GAT AAT GAA GAT CTT GTT TAT TGC TTG AAG GGA TGG AAT CAA2852 Val Asp Asp Asn Glu Asp Leu Val Tyr Cys Leu Lys Gly Trp Asn Gln 905910 915 GCT CTT TCA ATG TCC AAT GGT GGG GAC AAC CAT TGG GCT TTA TTT GCA2900 Ala Leu Ser Met Ser Asn Gly Gly Asp Asn His Trp Ala Leu Phe Ala 920925 930 AAA GCT GTG CTT GAC AGA ACC CGT CTT GCA CTT GCA AGC AAG GCA GAG2948 Lys Ala Val Leu Asp Arg Thr Arg Leu Ala Leu Ala Ser Lys Ala Glu 935940 945 TGG TAC CAT CAC TTA TTG CAG CCA TCT GCC GAA TAT CTA GGA TCA ATA2996 Trp Tyr His His Leu Leu Gln Pro Ser Ala Glu Tyr Leu Gly Ser Ile 950955 960 CTT GGG GTG GAC CAA TGG GCT TTG AAC ATA TTT ACT GAA GAA ATT ATA3044 Leu Gly Val Asp Gln Trp Ala Leu Asn Ile Phe Thr Glu Glu Ile Ile 965970 975 980 CGT GCT GGA TCA GCA GCT TCA TTA TCC TCT CTT CTT AAT AGA CTCGAT 3092 Arg Ala Gly Ser Ala Ala Ser Leu Ser Ser Leu Leu Asn Arg Leu Asp985 990 995 CCC GTG CTT CGG AAA ACT GCA AAT CTA GGA AGT TGG CAG ATT ATCAGT 3140 Pro Val Leu Arg Lys Thr Ala Asn Leu Gly Ser Trp Gln Ile Ile Ser1000 1005 1010 CCA GTT GAA GCC GTT GGA TAT GTT GTC GTT GTG GAT GAG TTGCTT TCA 3188 Pro Val Glu Ala Val Gly Tyr Val Val Val Val Asp Glu Leu LeuSer 1015 1020 1025 GTT CAG AAT GAA ATC TAC GAG AAG CCC ACG ATC TTA GTAGCA AAA TCT 3236 Val Gln Asn Glu Ile Tyr Glu Lys Pro Thr Ile Leu Val AlaLys Ser 1030 1035 1040 GTT AAA GGA GAG GAG GAA ATT CCT GAT GGT GCT GTTGCC CTG ATA ACA 3284 Val Lys Gly Glu Glu Glu Ile Pro Asp Gly Ala Val AlaLeu Ile Thr 1045 1050 1055 1060 CCA GAC ATG CCA GAT GTT CTT TCA CAT GTTTCT GTT CGA GCT AGA AAT 3332 Pro Asp Met Pro Asp Val Leu Ser His Val SerVal Arg Ala Arg Asn 1065 1070 1075 GGG AAG GTT TGC TTT GCT ACA TGC TTTGAT CCC AAT ATA TTG GCT GAC 3380 Gly Lys Val Cys Phe Ala Thr Cys Phe AspPro Asn Ile Leu Ala Asp 1080 1085 1090 CTC CAA GCA AAG GAA GGA AGG ATTTTG CTC TTA AAG CCT ACA CCT TCA 3428 Leu Gln Ala Lys Glu Gly Arg Ile LeuLeu Leu Lys Pro Thr Pro Ser 1095 1100 1105 GAC ATA ATC TAT AGT GAG GTGAAT GAG ATT GAG CTC CAA AGT TCA AGT 3476 Asp Ile Ile Tyr Ser Glu Val AsnGlu Ile Glu Leu Gln Ser Ser Ser 1110 1115 1120 AAC TTG GTA GAA GCT GAAACT TCA GCA ACA CTT AGA TTG GTG AAA AAG 3524 Asn Leu Val Glu Ala Glu ThrSer Ala Thr Leu Arg Leu Val Lys Lys 1125 1130 1135 1140 CAA TTT GGT GGTTGT TAC GCA ATA TCA GCA GAT GAA TTC ACA AGT GAA 3572 Gln Phe Gly Gly CysTyr Ala Ile Ser Ala Asp Glu Phe Thr Ser Glu 1145 1150 1155 ATG GTT GGAGCT AAA TCA CGT AAT ATT GCA TAT CTG AAA GGA AAA GTG 3620 Met Val Gly AlaLys Ser Arg Asn Ile Ala Tyr Leu Lys Gly Lys Val 1160 1165 1170 CCT TCCTCG GTG GGA ATT CCT ACG TCA GTA GCT CTT CCA TTT GGA GTC 3668 Pro Ser SerVal Gly Ile Pro Thr Ser Val Ala Leu Pro Phe Gly Val 1175 1180 1185 TTTGAG AAA GTA CTT TCA GAC GAC ATA AAT CAG GGA GTG GCA AAA GAG 3716 Phe GluLys Val Leu Ser Asp Asp Ile Asn Gln Gly Val Ala Lys Glu 1190 1195 1200TTG CAA ATT CTG ATG AAA AAA CTA TCT GAA GGA GAC TTC AGC GCT CTT 3764 LeuGln Ile Leu Met Lys Lys Leu Ser Glu Gly Asp Phe Ser Ala Leu 1205 12101215 1220 GGT GAA ATT CGC ACA ACG GTT TTA GAT CTT TCA GCA CCA GCT CAATTG 3812 Gly Glu Ile Arg Thr Thr Val Leu Asp Leu Ser Ala Pro Ala Gln Leu1225 1230 1235 GTC AAA GAG CTG AAG GAG AAG ATG CAG GGT TCT GGC ATG CCTTGG CCT 3860 Val Lys Glu Leu Lys Glu Lys Met Gln Gly Ser Gly Met Pro TrpPro 1240 1245 1250 GGT GAT GAA GGT CCA AAG CGG TGG GAA CAA GCA TGG ATGGCC ATA AAA 3908 Gly Asp Glu Gly Pro Lys Arg Trp Glu Gln Ala Trp Met AlaIle Lys 1255 1260 1265 AAG GTG TGG GCT TCA AAA TGG AAT GAG AGA GCA TACTTC AGC ACA AGG 3956 Lys Val Trp Ala Ser Lys Trp Asn Glu Arg Ala Tyr PheSer Thr Arg 1270 1275 1280 AAG GTG AAA CTG GAT CAT GAC TAT CTG TGC ATGGCT GTC CTT GTT CAA 4004 Lys Val Lys Leu Asp His Asp Tyr Leu Cys Met AlaVal Leu Val Gln 1285 1290 1295 1300 GAA ATA ATA AAT GCT GAT TAT GCA TTTGTC ATT CAC ACA ACC AAC CCA 4052 Glu Ile Ile Asn Ala Asp Tyr Ala Phe ValIle His Thr Thr Asn Pro 1305 1310 1315 TCT TCC GGA GAC GAC TCA GAA ATATAT GCC GAG GTG GTC AGG GGC CTT 4100 Ser Ser Gly Asp Asp Ser Glu Ile TyrAla Glu Val Val Arg Gly Leu 1320 1325 1330 GGG GAA ACA CTT GTT GGA GCTTAT CCA GGA CGT GCT TTG AGT TTT ATC 4148 Gly Glu Thr Leu Val Gly Ala TyrPro Gly Arg Ala Leu Ser Phe Ile 1335 1340 1345 TGC AAG AAA AAG GAT CTCAAC TCT CCT CAA GTG TTA GGT TAC CCA AGC 4196 Cys Lys Lys Lys Asp Leu AsnSer Pro Gln Val Leu Gly Tyr Pro Ser 1350 1355 1360 AAA CCG ATC GGC CTTTTC ATA AAA AGA TCT ATC ATC TTC CGA TCT GAT 4244 Lys Pro Ile Gly Leu PheIle Lys Arg Ser Ile Ile Phe Arg Ser Asp 1365 1370 1375 1380 TCC AAT GGGGAA GAT TTG GAA GGT TAT GCC GGT GCT GGC CTC TAC GAC 4292 Ser Asn Gly GluAsp Leu Glu Gly Tyr Ala Gly Ala Gly Leu Tyr Asp 1385 1390 1395 AGT GTACCA ATG GAT GAG GAG GAA AAA GTT GTA ATT GAT TAC TCT TCC 4340 Ser Val ProMet Asp Glu Glu Glu Lys Val Val Ile Asp Tyr Ser Ser 1400 1405 1410 GACCCA TTG ATA ACT GAT GGT AAC TTC CGC CAG ACA ATC CTG TCC AAC 4388 Asp ProLeu Ile Thr Asp Gly Asn Phe Arg Gln Thr Ile Leu Ser Asn 1415 1420 1425ATT GCT CGT GCT GGA CAT GCT ATC GAG GAG CTA TAT GGC TCT CCT CAA 4436 IleAla Arg Ala Gly His Ala Ile Glu Glu Leu Tyr Gly Ser Pro Gln 1430 14351440 GAC ATT GAG GGT GTA GTG AGG GAT GGA AAG ATT TAT GTC GTT CAG ACA4484 Asp Ile Glu Gly Val Val Arg Asp Gly Lys Ile Tyr Val Val Gln Thr1445 1450 1455 1460 AGA CCA CAG ATG T GATTATATTC TCGTTGTATG TTGTTCAGAGAAGACCACAG 4537 Arg Pro Gln Met ATGTGATCAT ATTCTCATTG TATCAGATCTGTGACCACTT ACCTGATACC TCCCATGAAG 4597 TTACCTGTAT GATTATACGT GATCCAAAGCCATCACATCA TGTTCACCTT CAGCTATTGG 4657 AGGAGAAGTG AGAAGTAGGA ATTGCAATATGAGGAATAAT AAGAAAAACT TTGTAAAAGC 4717 TAAATTAGCT GGGTATGATA TAGGGAGAAATGTGTAAACA TTGTACTATA TATAGTATAT 4777 ACACACGCAT TATGTATTGC ATTATGCACTGAATAATATC GCAGCATCAA AGAAGAAATC 4837 CTTTGGGTGG TTTCAAAAA 4856 1464amino acids amino acid linear protein unknown 2 Met Ser Asn Ser Leu GlyAsn Asn Leu Leu Tyr Gln Gly Phe Leu Thr 1 5 10 15 Ser Thr Val Leu GluHis Lys Ser Arg Ile Ser Pro Pro Cys Val Gly 20 25 30 Gly Asn Ser Leu PheGln Gln Gln Val Ile Ser Lys Ser Pro Leu Ser 35 40 45 Thr Glu Phe Arg GlyAsn Arg Leu Lys Val Gln Lys Lys Lys Ile Pro 50 55 60 Met Glu Lys Lys ArgAla Phe Ser Ser Ser Pro His Ala Val Leu Thr 65 70 75 80 Thr Asp Thr SerSer Glu Leu Ala Glu Lys Phe Ser Leu Gly Gly Asn 85 90 95 Ile Glu Leu GlnVal Asp Val Arg Pro Pro Thr Ser Gly Asp Val Ser 100 105 110 Phe Val AspPhe Gln Val Thr Asn Gly Ser Asp Lys Leu Phe Leu His 115 120 125 Trp GlyAla Val Lys Phe Gly Lys Glu Thr Trp Ser Leu Pro Asn Asp 130 135 140 ArgPro Asp Gly Thr Lys Val Tyr Lys Asn Lys Ala Leu Arg Thr Pro 145 150 155160 Phe Val Lys Ser Gly Ser Asn Ser Ile Leu Arg Leu Glu Ile Arg Asp 165170 175 Thr Ala Ile Glu Ala Ile Glu Phe Leu Ile Tyr Asp Glu Ala His Asp180 185 190 Lys Trp Ile Lys Asn Asn Gly Gly Asn Phe Arg Val Lys Leu SerArg 195 200 205 Lys Glu Ile Arg Gly Pro Asp Val Ser Val Pro Glu Glu LeuVal Gln 210 215 220 Ile Gln Ser Tyr Leu Arg Trp Glu Arg Lys Gly Lys GlnAsn Tyr Pro 225 230 235 240 Pro Glu Lys Glu Lys Glu Glu Tyr Glu Ala AlaArg Thr Val Leu Gln 245 250 255 Glu Glu Ile Ala Arg Gly Ala Ser Ile GlnAsp Ile Arg Ala Arg Leu 260 265 270 Thr Lys Thr Asn Asp Lys Ser Gln SerLys Glu Glu Pro Leu His Val 275 280 285 Thr Lys Ser Asp Ile Pro Asp AspLeu Ala Gln Ala Gln Ala Tyr Ile 290 295 300 Arg Trp Glu Lys Ala Gly LysPro Asn Tyr Pro Pro Glu Lys Gln Ile 305 310 315 320 Glu Glu Leu Glu GluAla Arg Arg Glu Leu Gln Leu Glu Leu Glu Lys 325 330 335 Gly Ile Thr LeuAsp Glu Leu Arg Lys Thr Ile Thr Lys Gly Glu Ile 340 345 350 Lys Thr LysVal Glu Lys His Leu Lys Arg Ser Ser Phe Ala Val Glu 355 360 365 Arg IleGln Arg Lys Lys Arg Asp Phe Gly His Leu Ile Asn Lys Tyr 370 375 380 ThrSer Ser Pro Ala Val Gln Val Gln Lys Val Leu Glu Glu Pro Pro 385 390 395400 Ala Leu Ser Lys Ile Lys Leu Tyr Ala Lys Glu Lys Glu Glu Gln Ile 405410 415 Asp Asp Pro Ile Leu Asn Lys Lys Ile Phe Lys Val Asp Asp Gly Glu420 425 430 Leu Leu Val Leu Val Ala Lys Ser Ser Gly Lys Thr Lys Val HisLeu 435 440 445 Ala Thr Asp Leu Asn Gln Pro Ile Thr Leu His Trp Ala LeuSer Lys 450 455 460 Ser Pro Gly Glu Trp Met Val Pro Pro Ser Ser Ile LeuPro Pro Gly 465 470 475 480 Ser Ile Ile Leu Asp Lys Ala Ala Glu Thr ProPhe Ser Ala Ser Ser 485 490 495 Ser Asp Gly Leu Thr Ser Lys Val Gln SerLeu Asp Ile Val Ile Glu 500 505 510 Asp Gly Asn Phe Val Gly Met Pro PheVal Leu Leu Ser Gly Glu Lys 515 520 525 Trp Ile Lys Asn Gln Gly Ser AspPhe Tyr Val Gly Phe Ser Ala Ala 530 535 540 Ser Lys Leu Ala Leu Lys AlaAla Gly Asp Gly Ser Gly Thr Ala Lys 545 550 555 560 Ser Leu Leu Asp LysIle Ala Asp Met Glu Ser Glu Ala Gln Lys Ser 565 570 575 Phe Met His ArgPhe Asn Ile Ala Ala Asp Leu Ile Glu Asp Ala Thr 580 585 590 Ser Ala GlyGlu Leu Gly Phe Ala Gly Ile Leu Val Trp Met Arg Phe 595 600 605 Met AlaThr Arg Gln Leu Ile Trp Asn Lys Asn Tyr Asn Val Lys Pro 610 615 620 ArgGlu Ile Ser Lys Ala Gln Asp Arg Leu Thr Asp Leu Leu Gln Asn 625 630 635640 Ala Phe Thr Ser His Pro Gln Tyr Arg Glu Ile Leu Arg Met Ile Met 645650 655 Ser Thr Val Gly Arg Gly Gly Glu Gly Asp Val Gly Gln Arg Ile Arg660 665 670 Asp Glu Ile Leu Val Ile Gln Arg Asn Asn Asp Cys Lys Gly GlyMet 675 680 685 Met Gln Glu Trp His Gln Lys Leu His Asn Asn Thr Ser ProAsp Asp 690 695 700 Val Val Ile Cys Gln Ala Leu Ile Asp Tyr Ile Lys SerAsp Phe Asp 705 710 715 720 Leu Gly Val Tyr Trp Lys Thr Leu Asn Glu AsnGly Ile Thr Lys Glu 725 730 735 Arg Leu Leu Ser Tyr Asp Arg Ala Ile HisSer Glu Pro Asn Phe Arg 740 745 750 Gly Asp Gln Lys Gly Gly Leu Leu ArgAsp Leu Gly His Tyr Met Arg 755 760 765 Thr Leu Lys Ala Val His Ser GlyAla Asp Leu Glu Ser Ala Ile Ala 770 775 780 Asn Cys Met Gly Tyr Lys ThrGlu Gly Glu Gly Phe Met Val Gly Val 785 790 795 800 Gln Ile Asn Pro ValSer Gly Leu Pro Ser Gly Phe Gln Asp Leu Leu 805 810 815 His Phe Val LeuAsp His Val Glu Asp Lys Asn Val Glu Thr Leu Leu 820 825 830 Glu Arg LeuLeu Glu Ala Arg Glu Glu Leu Arg Pro Leu Leu Leu Lys 835 840 845 Pro AsnAsn Arg Leu Lys Asp Leu Leu Phe Leu Asp Ile Ala Leu Asp 850 855 860 SerThr Val Arg Thr Ala Val Glu Arg Gly Tyr Glu Glu Leu Asn Asn 865 870 875880 Ala Asn Pro Glu Lys Ile Met Tyr Phe Ile Ser Leu Val Leu Glu Asn 885890 895 Leu Ala Leu Ser Val Asp Asp Asn Glu Asp Leu Val Tyr Cys Leu Lys900 905 910 Gly Trp Asn Gln Ala Leu Ser Met Ser Asn Gly Gly Asp Asn HisTrp 915 920 925 Ala Leu Phe Ala Lys Ala Val Leu Asp Arg Thr Arg Leu AlaLeu Ala 930 935 940 Ser Lys Ala Glu Trp Tyr His His Leu Leu Gln Pro SerAla Glu Tyr 945 950 955 960 Leu Gly Ser Ile Leu Gly Val Asp Gln Trp AlaLeu Asn Ile Phe Thr 965 970 975 Glu Glu Ile Ile Arg Ala Gly Ser Ala AlaSer Leu Ser Ser Leu Leu 980 985 990 Asn Arg Leu Asp Pro Val Leu Arg LysThr Ala Asn Leu Gly Ser Trp 995 1000 1005 Gln Ile Ile Ser Pro Val GluAla Val Gly Tyr Val Val Val Val Asp 1010 1015 1020 Glu Leu Leu Ser ValGln Asn Glu Ile Tyr Glu Lys Pro Thr Ile Leu 1025 1030 1035 1040 Val AlaLys Ser Val Lys Gly Glu Glu Glu Ile Pro Asp Gly Ala Val 1045 1050 1055Ala Leu Ile Thr Pro Asp Met Pro Asp Val Leu Ser His Val Ser Val 10601065 1070 Arg Ala Arg Asn Gly Lys Val Cys Phe Ala Thr Cys Phe Asp ProAsn 1075 1080 1085 Ile Leu Ala Asp Leu Gln Ala Lys Glu Gly Arg Ile LeuLeu Leu Lys 1090 1095 1100 Pro Thr Pro Ser Asp Ile Ile Tyr Ser Glu ValAsn Glu Ile Glu Leu 1105 1110 1115 1120 Gln Ser Ser Ser Asn Leu Val GluAla Glu Thr Ser Ala Thr Leu Arg 1125 1130 1135 Leu Val Lys Lys Gln PheGly Gly Cys Tyr Ala Ile Ser Ala Asp Glu 1140 1145 1150 Phe Thr Ser GluMet Val Gly Ala Lys Ser Arg Asn Ile Ala Tyr Leu 1155 1160 1165 Lys GlyLys Val Pro Ser Ser Val Gly Ile Pro Thr Ser Val Ala Leu 1170 1175 1180Pro Phe Gly Val Phe Glu Lys Val Leu Ser Asp Asp Ile Asn Gln Gly 11851190 1195 1200 Val Ala Lys Glu Leu Gln Ile Leu Met Lys Lys Leu Ser GluGly Asp 1205 1210 1215 Phe Ser Ala Leu Gly Glu Ile Arg Thr Thr Val LeuAsp Leu Ser Ala 1220 1225 1230 Pro Ala Gln Leu Val Lys Glu Leu Lys GluLys Met Gln Gly Ser Gly 1235 1240 1245 Met Pro Trp Pro Gly Asp Glu GlyPro Lys Arg Trp Glu Gln Ala Trp 1250 1255 1260 Met Ala Ile Lys Lys ValTrp Ala Ser Lys Trp Asn Glu Arg Ala Tyr 1265 1270 1275 1280 Phe Ser ThrArg Lys Val Lys Leu Asp His Asp Tyr Leu Cys Met Ala 1285 1290 1295 ValLeu Val Gln Glu Ile Ile Asn Ala Asp Tyr Ala Phe Val Ile His 1300 13051310 Thr Thr Asn Pro Ser Ser Gly Asp Asp Ser Glu Ile Tyr Ala Glu Val1315 1320 1325 Val Arg Gly Leu Gly Glu Thr Leu Val Gly Ala Tyr Pro GlyArg Ala 1330 1335 1340 Leu Ser Phe Ile Cys Lys Lys Lys Asp Leu Asn SerPro Gln Val Leu 1345 1350 1355 1360 Gly Tyr Pro Ser Lys Pro Ile Gly LeuPhe Ile Lys Arg Ser Ile Ile 1365 1370 1375 Phe Arg Ser Asp Ser Asn GlyGlu Asp Leu Glu Gly Tyr Ala Gly Ala 1380 1385 1390 Gly Leu Tyr Asp SerVal Pro Met Asp Glu Glu Glu Lys Val Val Ile 1395 1400 1405 Asp Tyr SerSer Asp Pro Leu Ile Thr Asp Gly Asn Phe Arg Gln Thr 1410 1415 1420 IleLeu Ser Asn Ile Ala Arg Ala Gly His Ala Ile Glu Glu Leu Tyr 1425 14301435 1440 Gly Ser Pro Gln Asp Ile Glu Gly Val Val Arg Asp Gly Lys IleTyr 1445 1450 1455 Val Val Gln Thr Arg Pro Gln Met 1460 1918 base pairsnucleotide single linear cDNA to mRNA Solanum tuberosum C.V. Desiree CDS1..1555 3 GCA GAG TGG TAC CAT CAC TTA TTG CAG CCA TCT GCC GAA TAT CTAGGA 48 Ala Glu Trp Tyr His His Leu Leu Gln Pro Ser Ala Glu Tyr Leu Gly 15 10 15 TCA ATA CTT GGG GTG GAC CAA TGG GCT TTG AAC ATA TTT ACT GAA GAA96 Ser Ile Leu Gly Val Asp Gln Trp Ala Leu Asn Ile Phe Thr Glu Glu 20 2530 ATT ATA CGT GCT GGA TCA GCA GCT TCA TTA TCC TCT CTT CTT AAT AGA 144Ile Ile Arg Ala Gly Ser Ala Ala Ser Leu Ser Ser Leu Leu Asn Arg 35 40 45CTC GAT CCC GTG CTT CGG AAA ACT GCA AAT CTA GGA AGT TGG CAG ATT 192 LeuAsp Pro Val Leu Arg Lys Thr Ala Asn Leu Gly Ser Trp Gln Ile 50 55 60 ATCAGT CCA GTT GAA GCC GTT GGA TAT GTT GTC GTT GTG GAT GAG TTG 240 Ile SerPro Val Glu Ala Val Gly Tyr Val Val Val Val Asp Glu Leu 65 70 75 80 CTTTCA GTT CAG AAT GAA ATC TAC GAG AAG CCC ACG ATC TTA GTA GCA 288 Leu SerVal Gln Asn Glu Ile Tyr Glu Lys Pro Thr Ile Leu Val Ala 85 90 95 AAA TCTGTT AAA GGA GAG GAG GAA ATT CCT GAT GGT GCT GTT GCC CTG 336 Lys Ser ValLys Gly Glu Glu Glu Ile Pro Asp Gly Ala Val Ala Leu 100 105 110 ATA ACACCA GAC ATG CCA GAT GTT CTT TCA CAT GTT TCT GTT CGA GCT 384 Ile Thr ProAsp Met Pro Asp Val Leu Ser His Val Ser Val Arg Ala 115 120 125 AGA AATGGG AAG GTT TGC TTT GCT ACA TGC TTT GAT CCC AAT ATA TTG 432 Arg Asn GlyLys Val Cys Phe Ala Thr Cys Phe Asp Pro Asn Ile Leu 130 135 140 GCT GACCTC CAA GCA AAG GAA GGA AGG ATT TTG CTC TTA AAG CCT ACA 480 Ala Asp LeuGln Ala Lys Glu Gly Arg Ile Leu Leu Leu Lys Pro Thr 145 150 155 160 CCTTCA GAC ATA ATC TAT AGT GAG GTG AAT GAG ATT GAG CTC CAA AGT 528 Pro SerAsp Ile Ile Tyr Ser Glu Val Asn Glu Ile Glu Leu Gln Ser 165 170 175 TCAAGT AAC TTG GTA GAA GCT GAA ACT TCA GCA ACA CTT AGA TTG GTG 576 Ser SerAsn Leu Val Glu Ala Glu Thr Ser Ala Thr Leu Arg Leu Val 180 185 190 AAAAAG CAA TTT GGT GGT TGT TAC GCA ATA TCA GCA GAT GAA TTC ACA 624 Lys LysGln Phe Gly Gly Cys Tyr Ala Ile Ser Ala Asp Glu Phe Thr 195 200 205 AGTGAA ATG GTT GGA GCT AAA TCA CGT AAT ATT GCA TAT CTG AAA GGA 672 Ser GluMet Val Gly Ala Lys Ser Arg Asn Ile Ala Tyr Leu Lys Gly 210 215 220 AAAGTG CCT TCC TCG GTG GGA ATT CCT ACG TCA GTA GCT CTT CCA TTT 720 Lys ValPro Ser Ser Val Gly Ile Pro Thr Ser Val Ala Leu Pro Phe 225 230 235 240GGA GTC TTT GAG AAA GTA CTT TCA GAC GAC ATA AAT CAG GGA GTG GCA 768 GlyVal Phe Glu Lys Val Leu Ser Asp Asp Ile Asn Gln Gly Val Ala 245 250 255AAA GAG TTG CAA ATT CTG ACA AAA AAA CTA TCT GAA GGA GAC TTT AGC 816 LysGlu Leu Gln Ile Leu Thr Lys Lys Leu Ser Glu Gly Asp Phe Ser 260 265 270GCT CTT GGT GAA ATT CGC ACA ACG GTT TTA GAT CTT TCG ACA CCA GCT 864 AlaLeu Gly Glu Ile Arg Thr Thr Val Leu Asp Leu Ser Thr Pro Ala 275 280 285CAA TTG GTC AAA GAG CTG AAG GAG AAG ATG CAG GGT TCT GGC ATG CCT 912 GlnLeu Val Lys Glu Leu Lys Glu Lys Met Gln Gly Ser Gly Met Pro 290 295 300TGG CCT GGT GAT GAA GGT CCA AAG CGG TGG GAA CAA GCA TGG ATG GCC 960 TrpPro Gly Asp Glu Gly Pro Lys Arg Trp Glu Gln Ala Trp Met Ala 305 310 315320 ATA AAA AAG GTG TGG GCT TCA AAA TGG AAT GAG AGA GCA TAC TTC AGC 1008Ile Lys Lys Val Trp Ala Ser Lys Trp Asn Glu Arg Ala Tyr Phe Ser 325 330335 ACA AGG AAG GTG AAA CTG GAT CAT GAC TAT CTG TGC ATG GCT GTC CTT 1056Thr Arg Lys Val Lys Leu Asp His Asp Tyr Leu Cys Met Ala Val Leu 340 345350 GTT CAA GAA ATA ATA AAT GCT GAT TAT GCA TTT GTC ATT CAC ACA ACC 1104Val Gln Glu Ile Ile Asn Ala Asp Tyr Ala Phe Val Ile His Thr Thr 355 360365 AAC CCA TCT TCC GGA GAC GAC TCA GAA ATA TAT GCC GAG GTG GTC AGG 1152Asn Pro Ser Ser Gly Asp Asp Ser Glu Ile Tyr Ala Glu Val Val Arg 370 375380 GGC CTT GGG GAA ACA CTT GTT GGA GCT TAT CCA GGA CGT GCT TTG AGT 1200Gly Leu Gly Glu Thr Leu Val Gly Ala Tyr Pro Gly Arg Ala Leu Ser 385 390395 400 TTT ATC TGC AAG AAA AAG GAT CTC AAC TCT CCT CAA GTG TTA GGT TAC1248 Phe Ile Cys Lys Lys Lys Asp Leu Asn Ser Pro Gln Val Leu Gly Tyr 405410 415 CCA AGC AAA CCG ATC GGC CTT TTC ATA AAA AGA TCT ATC ATC TTC CGA1296 Pro Ser Lys Pro Ile Gly Leu Phe Ile Lys Arg Ser Ile Ile Phe Arg 420425 430 TCT GAT TCC AAT GGG GAA GAT TTG GAA GGT TAT GCC GGT GCT GGC CTC1344 Ser Asp Ser Asn Gly Glu Asp Leu Glu Gly Tyr Ala Gly Ala Gly Leu 435440 445 TAC GAC AGT GTA CCA ATG GAT GAG GAG GAA AAA GTT GTA ATT GAT TAC1392 Tyr Asp Ser Val Pro Met Asp Glu Glu Glu Lys Val Val Ile Asp Tyr 450455 460 TCT TCC GAC CCA TTG ATA ACT GAT GGT AAC TTC CGC CAG ACA ATC CTG1440 Ser Ser Asp Pro Leu Ile Thr Asp Gly Asn Phe Arg Gln Thr Ile Leu 465470 475 480 TCC AAC ATT GCT CGT GCT GGA CAT GCT ATC GAG GAG CTA TAT GGCTCT 1488 Ser Asn Ile Ala Arg Ala Gly His Ala Ile Glu Glu Leu Tyr Gly Ser485 490 495 CCT CAA GAC ATT GAG GGT GTA GTG AGG GAT GGA AAG ATT TAT GTCGTT 1536 Pro Gln Asp Ile Glu Gly Val Val Arg Asp Gly Lys Ile Tyr Val Val500 505 510 CAG ACA AGA CCA CAG ATG T GATTATATTC TCGTTGTATG TTGTTCAGAG1585 Gln Thr Arg Pro Gln Met 515 AAGACCACAG ATGTGATCAT ATTCTCATTGTATCAGATCT GTGACCACTT ACCTGATACC 1645 TCCCATGAAG TTACCTGTAT GATTATACGTGATCCAAAGC CATCACATCA TGTTCACCTT 1705 CAGCTATTGG AGGAGAAGTG AGAAGTAGGAATTGCAATAT GAGGAATAAT AAGAAAAACT 1765 TTGTAAAAGC TAAATTAGCT GGGTATGATATAGGGAGAAA TGTGTAAACA TTGTACTATA 1825 TATAGTATAT ACACACGCAT TATGTATTGCATTATGCACT GAATAATATC GCAGCATCAA 1885 AGAAGAAATC CTTTGGGTGG TTTCAAAAAAAAA 1918 518 amino acids amino acid linear protein unknown 4 Ala Glu TrpTyr His His Leu Leu Gln Pro Ser Ala Glu Tyr Leu Gly 1 5 10 15 Ser IleLeu Gly Val Asp Gln Trp Ala Leu Asn Ile Phe Thr Glu Glu 20 25 30 Ile IleArg Ala Gly Ser Ala Ala Ser Leu Ser Ser Leu Leu Asn Arg 35 40 45 Leu AspPro Val Leu Arg Lys Thr Ala Asn Leu Gly Ser Trp Gln Ile 50 55 60 Ile SerPro Val Glu Ala Val Gly Tyr Val Val Val Val Asp Glu Leu 65 70 75 80 LeuSer Val Gln Asn Glu Ile Tyr Glu Lys Pro Thr Ile Leu Val Ala 85 90 95 LysSer Val Lys Gly Glu Glu Glu Ile Pro Asp Gly Ala Val Ala Leu 100 105 110Ile Thr Pro Asp Met Pro Asp Val Leu Ser His Val Ser Val Arg Ala 115 120125 Arg Asn Gly Lys Val Cys Phe Ala Thr Cys Phe Asp Pro Asn Ile Leu 130135 140 Ala Asp Leu Gln Ala Lys Glu Gly Arg Ile Leu Leu Leu Lys Pro Thr145 150 155 160 Pro Ser Asp Ile Ile Tyr Ser Glu Val Asn Glu Ile Glu LeuGln Ser 165 170 175 Ser Ser Asn Leu Val Glu Ala Glu Thr Ser Ala Thr LeuArg Leu Val 180 185 190 Lys Lys Gln Phe Gly Gly Cys Tyr Ala Ile Ser AlaAsp Glu Phe Thr 195 200 205 Ser Glu Met Val Gly Ala Lys Ser Arg Asn IleAla Tyr Leu Lys Gly 210 215 220 Lys Val Pro Ser Ser Val Gly Ile Pro ThrSer Val Ala Leu Pro Phe 225 230 235 240 Gly Val Phe Glu Lys Val Leu SerAsp Asp Ile Asn Gln Gly Val Ala 245 250 255 Lys Glu Leu Gln Ile Leu ThrLys Lys Leu Ser Glu Gly Asp Phe Ser 260 265 270 Ala Leu Gly Glu Ile ArgThr Thr Val Leu Asp Leu Ser Thr Pro Ala 275 280 285 Gln Leu Val Lys GluLeu Lys Glu Lys Met Gln Gly Ser Gly Met Pro 290 295 300 Trp Pro Gly AspGlu Gly Pro Lys Arg Trp Glu Gln Ala Trp Met Ala 305 310 315 320 Ile LysLys Val Trp Ala Ser Lys Trp Asn Glu Arg Ala Tyr Phe Ser 325 330 335 ThrArg Lys Val Lys Leu Asp His Asp Tyr Leu Cys Met Ala Val Leu 340 345 350Val Gln Glu Ile Ile Asn Ala Asp Tyr Ala Phe Val Ile His Thr Thr 355 360365 Asn Pro Ser Ser Gly Asp Asp Ser Glu Ile Tyr Ala Glu Val Val Arg 370375 380 Gly Leu Gly Glu Thr Leu Val Gly Ala Tyr Pro Gly Arg Ala Leu Ser385 390 395 400 Phe Ile Cys Lys Lys Lys Asp Leu Asn Ser Pro Gln Val LeuGly Tyr 405 410 415 Pro Ser Lys Pro Ile Gly Leu Phe Ile Lys Arg Ser IleIle Phe Arg 420 425 430 Ser Asp Ser Asn Gly Glu Asp Leu Glu Gly Tyr AlaGly Ala Gly Leu 435 440 445 Tyr Asp Ser Val Pro Met Asp Glu Glu Glu LysVal Val Ile Asp Tyr 450 455 460 Ser Ser Asp Pro Leu Ile Thr Asp Gly AsnPhe Arg Gln Thr Ile Leu 465 470 475 480 Ser Asn Ile Ala Arg Ala Gly HisAla Ile Glu Glu Leu Tyr Gly Ser 485 490 495 Pro Gln Asp Ile Glu Gly ValVal Arg Asp Gly Lys Ile Tyr Val Val 500 505 510 Gln Thr Arg Pro Gln Met515

What is claimed is:
 1. An isolated nucleic acid molecule encoding aprotein that is present in plant cells in starch granule-bound form aswell as in soluble form and that is involved in the phosphorylation ofstarch when expressed in plants and/or that increases thephosphorylation of glycogen when expressed in E. coli, selected from thegroup consisting of: (a) a nucleic acid molecule comprising a nucleotidesequence that encodes a protein having the amino acid sequence of SEQ IDNO: 2; (b) a nucleic acid molecule comprising the coding region of thenucleotide sequence of SEQ ID NO: 1; (c) a nucleic acid molecule thathybridizes to the nucleic acid molecule of (a) or (b) under stringentconditions; (d) a nucleic acid molecule the nucleotide sequence of whichis degenerate as a result of the genetic code to a nucleotide sequenceof the nucleic acid molecule of (a), (b) or (c); and (e) a fragment orallelic variant of the nucleic acid molecule of (a), (b), (c) or (d),wherein said fragment is of sufficient length to encode a protein thatis present in plant cells in starch granule-bound form as well as insoluble form and that is involved in the phosphorylation of starch whenexpressed in plants and/or that increases the phosphorylation ofglycogen when expressed in E. coli.
 2. An isolated nucleic acid moleculeencoding a protein that is present in plant cells in starchgranule-bound form as well as in soluble form and that is involved inthe phosphorylation of starch when expressed in plants and/or thatincreases the phosphorylation of glycogen when expressed in E. coli,wherein said nucleic acid molecule exhibits a sequence identity of atleast 60% to (a) a nucleic acid molecule comprising a nucleotidesequence that encodes a protein having the amino acid sequence of SEQ IDNO: 2; or (b) a nucleic acid molecule comprising the coding region ofthe nucleotide sequence of SEQ ID NO:
 1. 3. The nucleic acid moleculeaccording to claim 2, wherein said sequence identity is greater than80%.
 4. The nucleic acid molecule according to claim 2, wherein saidsequence identity is greater than 90%.
 5. A vector comprising thenucleic acid molecule according to any one of claims 1, 2, 3 or
 4. 6.The vector according to claim 5, wherein the nucleic acid molecule islinked to regulatory elements ensuring transcription in eukaryotic orprokaryotic cells.
 7. A host cell genetically modified with the nucleicacid molecule according to any one of claims 1, 2, 3 or 4 or with avector comprising said nucleic acid molecule.
 8. A transgenic plant celltransformed with the nucleic acid molecule according to any one ofclaims 1, 2, 3 or 4 that is linked in sense orientation to regulatoryDNA elements ensuring transcription in plant cells.
 9. A transgenicplant comprising the transgenic plant cell according to claim
 8. 10. Amethod for the production of a protein that is present in plant cells instarch granule-bound form as well as in soluble form and that isinvolved in the phosphorylation of starch when expressed in plantsand/or that increases the phosphorylation of glycogen when expressed inE. coli, comprising the steps of cultivating the host cell according toclaim 7 under conditions allowing for the expression of the protein andisolating the protein from the cells and/or the culture medium.
 11. Amethod for the production of a transgenic plant cell synthesizing amodified starch wherein the amount of protein encoded by the nucleicacid molecule according to any one of claims 1, 2, 3 or 4 is increasedin the transgenic plant cell when compared to a wildtype plant cell,comprising the steps of transforming a plant cell with said nucleic acidmolecule and expressing said nucleic acid molecule.
 12. A propagationmaterial of a transgenic plant comprising the plant cell according toclaim
 8. 13. A transgenic plant cell comprising the nucleic acidmolecule according to any one of claims 1, 2, 3 or 4, wherein saidnucleic acid molecule is introduced into said cell and wherein said cellsynthesizes a modified starch compared to starch from wild type cells,wherein the amount of a protein encoded by said nucleic acid molecule isincreased in the transgenic plant cell when compared to a wild typeplant cell.
 14. A transgenic plant cell comprising the nucleic acidmolecule according to any one of claims 1, 2, 3 or 4, wherein saidnucleic acid molecule is introduced into said cell and wherein said cellsynthesizes a starch with an increased phosphate content compared tostarch from wild type cells, wherein the amount of a protein encoded bysaid nucleic acid molecule is increased in the transgenic plant cellwhen compared to the wild type plant cell.
 15. The transgenic plantaccording to claim 9 which is a rye, barley, oat, wheat, rice, maize,pea, cassava or potato plant.